Tris-quaternary ammonium salts and methods for modulating neuronal nicotinic acetylcholine receptors

ABSTRACT

Provided are tris-quaternary ammonium compounds which are modulators of nicotinic acetylocholine receptors. Also provided are methods of using the compounds for modulating the function of a nicotinic acetylcholine receptor, and for the prevention and/or treatment of central nervous system disorders, substance use and/or abuse and or gastrointestinal tract disorders.

FIELD OF THE INVENTION

The invention relates to tris-quaternary ammonium salts and their use inmodulating nicotinic acetylcholine receptors.

BACKGROUND OF THE INVENTION

S(−)-nicotine (NIC) activates presynaptic and postsynaptic neuronalnicotinic receptors that evoke the release of neurotransmitters frompresynaptic terminals and that modulate the depolarization state of thepostsynaptic neuronal membrane, respectively. Thus, nicotine producesits effect by binding to a family of ligand-gated ion channels,stimulated by acetylcholine (ACh) or nicotine which causes the ionchannel to open and cations to flux with a resulting rapid (millisecond)depolarization of the target cell. Neuronal nicotinic receptors arecomposed of two types of subunits, α and β, and assemble as heteromericreceptors with the general stoichiometry of 2α and 3β or as homomericreceptors with 5α subunits. Nine subtypes of the α subunit (α2 to α10)and three subtypes of the β unit (β2 to β4) are found in the centralnervous system. The most common nicotinic receptor subtype in the brainis composed of two α4 and three 132 subunits, i.e., α4β2. These subunitsdisplay different, but overlapping, patterns of expression in the brain.Examples of heteromeric receptor subtypes include α4β2, α3β2, α3β4,α6β2, α4α5β2, α6α5β2, α4α6β2, α4β2β4, α3β2β4, and others. Thepredominant homomeric subtype includes α7, but other combinations havealso been proposed.

For the most part, the actual subunit compositions and stoichiometriesof nicotinic receptors in the brain remain to be elucidated. Thus,neuronal nicotinic receptor subtype diversity originates fromdifferences in the amino acid sequence at the subunit level and from themultiple combinations of assemblies of subunits into functional receptorproteins, which affords a wide diversity of pharmacological specificity.

In spite of the extensive diversity in neuronal nicotinic receptormessenger RNA expression, only a limited number of tools are availableto study the pharmacology of native receptors. Radioligands are used inmany studies. [³H]NIC appears to label the same sites in the brain as[³H]ACh. It has been estimated that over 90% of [³H]NIC binding in thebrain is due to association with the heteromeric receptor that iscomposed of α4 and β2 subunits. Also abundant in the central nervoussystem are the homomeric receptors labeled by [³H]methyllycaconitine(MLA), which has high affinity for the α7 nicotinic receptor subtype.Nicotinic receptor subtypes can be studied using functional assays, suchas NIC-evoked neurotransmitter release (e.g., [³H]dopamine (DA) release,[³H]norepinephrine (NE) release, [³H]serotonin (5-HT) release,[³H]gamma-aminobutyric acid (GABA) release and [³H]glutamate release)from superfused rat brain slices. Nicotinic receptors are located in thecell body and terminal areas of these neurotransmitter systems. NICfacilitates neurotransmitter release from nerve terminals.

The structural and functional diversity of central nervous systemnicotinic receptors has stimulated a great deal of interest indeveloping novel, subtype-selective agonists and/or antagonists. Some ofthese agonists are currently being evaluated in clinical trials forcognitive enhancement and neuroprotective effects, potentiallybeneficial for disease states such as Alzheimer's and Parkinson'sdisease.

SUMMARY OF INVENTION

In one embodiment, compounds corresponding to the following structureare provided.

-   The three side chains attached to the phenyl ring may be connected    to the 1, 2, and 3 positions; the 1, 2, and 4 positions; or the 1, 3    and 5 positions of the phenyl ring.-   The values for nil, m2 and m3 are each independently 0, 1, 2, 3, 4    or 5.-   The values for n1, n2, and n3 are each independently 1, 2, 3, 4 or    5.-   X^(1⊖), X^(2⊖), and X^(3⊖) are each independently an organic or    inorganic anion.-   L¹, L² and L³ are each independently chosen from the group    consisting of —CH₂—CH₂—, cis —CH═CH—, trans —CH═CH—, —C≡C—, —CH₂—S—,    —S—CH₂—, —Se—CH₂—, —CH₂—Se—, —CH₂—O—, —O—CH₂—, —CH₂—NH—, —NH—CH₂—,    —CH₂—NR— where R is a branched or straight chain alkyl group of one    to four carbons, —NR—CH₂— where R is a branched or straight chain    alkyl group of one to four carbons, —CH═N—, —N═CH—, and —N═N—.-   R¹, R², and R³ are each independently five or six membered nitrogen    containing rings as shown in formulas (IIA) and (IIB).

-   A¹ is carbon or nitrogen, provided that when A¹ joins a ring atom    with an unsaturated bond or is a nitrogen, R⁹ is absent, and when A¹    joins a ring atom an unsaturated bond and is a nitrogen, both R⁴ and    R⁹ are absent.-   A² is carbon or nitrogen, provided that when A² joins a ring atom an    unsaturated bond or is a nitrogen, R¹⁰ is absent, and when A² joins    a ring atom an unsaturated bond and is a nitrogen, both R⁵ and R¹⁰    are absent.-   A³ is carbon or nitrogen, provided that when A³ joins a ring atom an    unsaturated bond or is a nitrogen, R¹¹ is absent, and when A³ joins    a ring atom an unsaturated bond and is a nitrogen, both R⁶ and R¹¹    are absent.-   A⁴ is carbon or nitrogen; provided that when A⁴ joins a ring atom an    unsaturated bond or is a nitrogen, R¹² is absent, and when A⁴ joins    a ring atom an unsaturated bond and is a nitrogen, both R⁷ and R¹²    are absent.-   A⁵ is carbon or nitrogen, provided that when A⁵ joins a ring atom an    unsaturated bond or is a nitrogen, R¹³ is absent, and when A⁵ joins    a ring atom an unsaturated bond and is a nitrogen, both R⁸ and R¹³    are absent.-   A⁶ is carbon or nitrogen, provided that when A⁶ joins a ring atom an    unsaturated bond or is a nitrogen, R¹⁹ is absent, and when A⁶ joins    a ring atom an unsaturated bond and is a nitrogen, both R¹⁵ and R¹⁹    are absent.-   A⁷ is carbon or nitrogen, provided that when A⁷ joins a ring atom an    unsaturated bond or is a nitrogen, R²⁰ is absent, and when A⁷ joins    a ring atom an unsaturated bond and is a nitrogen, both R¹⁶ and R²⁰    are absent.-   A⁸ is carbon or nitrogen, provided that when A⁸ joins a ring atom an    unsaturated bond or is a nitrogen, R²¹ is absent, and when A⁸ joins    a ring atom an unsaturated bond and is a nitrogen, both R¹⁷ and R²¹    are absent.-   A⁹ is carbon or nitrogen, provided that when A⁹ joins a ring atom an    unsaturated bond or is a nitrogen, R²² is absent, and when A⁹ joins    a ring atom an unsaturated bond and is a nitrogen, both R¹⁸ and R²²    are absent.-   R¹⁴ or R²³ is absent when any of the bonds to the ammonium nitrogen    is unsaturated, and R¹⁴ or R²³ is a straight chain or branched alkyl    group of four carbons or fewer when all of the bonds to the ammonium    nitrogen are saturated.-   R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹³ or R¹⁵, R¹⁶, R¹⁷,    R¹⁸, R¹⁹, R²⁰, R²¹, and R²², when present, are each independently    selected from hydrogen; alkyl; substituted alkyl; cycloalkyl;    substituted cycloalkyl; alkenyl; substituted alkenyl; alkynyl;    substituted alkynyl; aryl; substituted aryl; alkylaryl; substituted    alkylaryl; arylalkyl; substituted arylalkyl; arylalkenyl;    substituted arylalkenyl; arylalkynyl; substituted arylalkynyl;    heterocyclic; substituted heterocyclic; halo; cyano; nitro; SOY¹,    SO₂Y¹, SO₂OY¹ or SO₂NHY¹, where Y¹ is selected from hydrogen, lower    alkyl, alkenyl, alkynyl or aryl, and where Y¹ is not hydrogen in    SOY¹ and if Y¹ is alkenyl or alkynyl, the site of unsaturation is    not conjugated with a heteroatom; COY², where Y² is selected from    hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted    cycloalkyl, alkoxy, alkenyl, substituted alkenyl, alkynyl,    substituted alkynyl, aryl, substituted aryl, alkylaryl, substituted    alkylaryl, arylalkyl, substituted arylalkyl, arylalkenyl,    substituted arylalkenyl, arylalkynyl, substituted arylalkynyl,    heterocyclic, or substituted heterocyclic, and where if Y² comprises    alkenyl or alkynyl, the site of unsaturation is not conjugated with    the carbonyl group; OY³, where Y³ is selected from hydrogen, alkyl,    substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,    substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted    aryl, alkylaryl, substituted alkylaryl, arylalkyl, substituted    arylalkyl, arylalkenyl, substituted arylalkenyl, arylalkynyl,    substituted arylalkynyl, acyl, substituted acyl, alkylsulfonyl,    arylsulfonyl, heterocyclic, or substituted heterocyclic, where if Y³    comprises alkenyl or alkynyl, the site of unsaturation is not    conjugated with the oxygen; NY⁴Y⁵, where Y⁴ and Y⁵ are each    independently selected from hydrogen, alkyl, substituted alkyl,    cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,    alkynyl, substituted alkynyl, aryl, substituted aryl, alkylaryl,    substituted alkylaryl, arylalkyl, substituted arylalkyl,    arylalkenyl, substituted arylalkenyl, arylalkynyl, substituted    arylalkynyl, acyl, substituted acyl, alkylsulfonyl, arylsulfonyl,    heterocyclic, or substituted heterocyclic, where if Y⁴ or Y⁵    comprises alkenyl or alkynyl, the site of unsaturation is not    conjugated with the nitrogen; SY⁶, where Y⁶ is selected from    hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted    cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted    alkynyl, aryl, substituted aryl, alkylaryl, substituted alkylaryl,    arylalkyl, substituted arylalkyl, arylalkenyl, substituted    arylalkenyl, arylalkynyl, substituted arylalkynyl, heterocyclic, or    substituted heterocyclic, and where if Y⁶ comprises alkenyl or    alkynyl, the site of unsaturation is not conjugated with the sulfur;    or R⁴ and R⁵ together with A¹ and A², or R⁵ and R⁶ together with A²    and A³, or R¹⁵ and R¹⁶ together with A⁶ and A⁷, or R¹⁶ and R¹⁷    together with A⁷ and A⁸ independently form a three to eight member    cyclolkane, substituted cycloalkane, cycloalkene, substituted    cycloalkene, aryl, substituted aryl, heterocycle with one to three    hetero atoms in the ring, or substituted heterocycle with one to    three hetero atoms in the ring.

In another embodiment, a composition is provided comprising apharmaceutically acceptable carrier and a compound as described above.

In another embodiment, a method is provided for selectively modulatingthe function of a nicotinic acetylcholine receptor comprisingadministering a therapeutically effective amount of a compound asdescribed above to a mammalian subject in need thereof.

In another embodiment, a method is provided for preventing and/ortreating a central nervous system associated disorder comprisingadministering a therapeutically effective amount of a compound asdescribed above to a mammalian subject in need thereof.

In another embodiment, a method is provided for preventing and/ortreating substance use and/or abuse comprising administering atherapeutically effective amount of a compound as described above to amammalian subject in need thereof.

In another embodiment, a method is provided for preventing and/ortreating gastrointestinal tract disorders comprising administering atherapeutically effective amount of a compound as described above to amammalian subject in need thereof.

Other methods, features and advantages of the present invention will beor become apparent to one with skill in the art upon examination of thefollowing detailed descriptions. It is intended that all such additionalmethods, features and advantages be included within this description, bewithin the scope of the present invention, and be protected by theaccompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows concentration-response curves for GZ551A in each foursubtypes of nicotinic receptor (α4β2, α3β2, α3β4 and α7) expressed inXenopus oocytes.

FIG. 2 shows concentration-response curves for GZ551B in each foursubtypes of nicotinic receptor (α4β2, α3β2, α3β4 and α7) expressed inXenopus oocytes.

FIG. 3 shows concentration-response curves for GZ558C in each foursubtypes of nicotinic receptor (α4β2, α3β2, α3β4 and α7) expressed inXenopus oocytes.

FIG. 4 shows the inhibitory response of the α7 nicotinic receptorsubtype expressed in Xenopus oocytes to 1.0 mM analog concentration for17 analogs co-applied with 60 mM acetylcholine (filled bars), as well asrecovery from analog-induced inhibition following washing of thepreparation (open bars).

DETAILED DESCRIPTION OF INVENTION

Before the present compositions and methods are described, it is to beunderstood that the invention is not limited to the particularmethodologies, protocols, assays, and reagents described, as these mayvary. It is also to be understood that the terminology used herein isintended to describe particular embodiments of the present invention,and is in no way intended to limit the scope of the present invention asset forth in the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural references unless thecontext clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. All publications citedherein are incorporated herein by reference in their entirety for thepurpose of describing and disclosing the methodologies, reagents, andtools reported in the publications that might be used in connection withthe invention. Nothing herein is to be construed as an admission thatthe invention is not entitled to antedate such disclosure by virtue ofprior invention.

The term “nicotinic acetylcholine receptor” refers to the endogenousacetylcholine receptor having binding sites for acetylcholine which alsobind to nicotine. The term “nicotinic acetylcholine receptor” includesthe term “neuronal nicotinic acetylcholine receptor.”

The terms “subtype of nicotinic acetylcholine receptor,” and “nicotinicacetylcholine receptor subtype” refer to various subunit combinations ofthe nicotinic acetylcholine receptor, and may refer to a particularhomomeric or heteromeric complex, or multiple homomeric or heteromericcomplexes.

The term “agonist” refers to a substance which interacts with a receptorand increases or prolongs a physiological response (i.e. activates thereceptor).

The term “partial agonist” refers to a substance which interacts withand activates a receptor to a lesser degree than an agonist.

The term “antagonist” refers to a substance which interacts with anddecreases the extent or duration of a physiological response of thatreceptor.

The terms “disorder,” “disease,” and “condition” are used inclusivelyand refer to any status deviating from normal.

The term “central nervous system associated disorders” includes anycognitive, neurological, and mental disorders causing aberrant orpathological neural signal transmission, such as disorders associatedwith the alteration of normal neurotransmitter release in the brain.

The term “lower alkyl” refers to straight or branched chain alkylradicals having in the range of 1 to 4 carbon atoms.

The term “alkyl” refers to straight or branched chain alkyl radicalshaving 1 to 19 carbon atoms, and “substituted alkyl” refers to alkylradicals further bearing one or more substituents including, but notlimited to, hydroxy, alkoxy (of a lower alkyl group), mercapto (of alower alkyl group), aryl, heterocyclic, halogen, trifluoromethyl, cyano,nitro, amino, carboxyl, carbamate, sulfonyl, and sulfonamide.

The term “cycloalkyl” refers to cyclic ring-containing moietiescontaining 3 to 8 carbon atoms, and “substituted cycloalkyl” refers tocycloalkyl moieties further bearing one or more substituents as setforth above.

The term “alkenyl” refers to straight or branched chain hydrocarbylgroups having at least one carbon-carbon double bond and having 2 to 19carbon atoms, and “substituted alkenyl” refers to alkenyl groups furtherbearing one or more substituents as set forth above.

The term “alkynyl” refers to straight or branched chain hydrocarbylmoieties having at least one carbon-carbon triple bond and having 2 to19 carbon atoms, and “substituted alkynyl” refers to alkynyl moietiesfurther bearing one or more substituents as set forth above.

The term “aryl” refers to aromatic groups having 6 to 24 carbon atoms,and “substituted aryl” refers to aryl groups further bearing one or moresubstituents as set forth above.

The term “alkylaryl” refers to alkyl-substituted aryl groups, and“substituted alkylaryl” refers to alkylaryl groups further bearing oneor more substituents as set forth above.

The term “arylalkyl” refers to aryl-substituted alkyl groups, and“substituted arylalkyl” refers to arylalkyl groups further bearing oneor more substituents as set forth above.

The term “arylalkenyl” refers to aryl-substituted alkenyl groups, and“substituted arylalkenyl” refers to arylalkenyl groups further bearingone or more substituents as set forth above.

The term “arylalkynyl” refers to aryl-substituted alkynyl groups, and“substituted arylalkynyl” refers to arylalkynyl groups further bearingone or more substituents as set forth above.

The term “heterocyclic” refers to cyclic moieties containing one or moreheteroatoms as part of the ring structure and having 3 to 24 carbonatoms, and “substituted heterocyclic” refers to heterocyclic moietiesfurther bearing one or more substituents as set forth above.

The term “acyl” refers to alkyl-carbonyl groups, and “substituted acyl”refers to acyl groups further bearing one or more substituents as setforth above.

The term “halogen” refers to fluoride, chloride, bromide or iodidegroups.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g. substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,etc.) are not intended for inclusion herein. In such cases, the maximumnumber of such substituents is three. That is to say that each of theabove definitions is constrained by a limitation that, for example,substituted aryl groups are limited to -substituted aryl-(substitutedaryl)-substituted aryl.

Compounds of the present invention are tris-quaternary ammonium saltscorresponding to Formula (I):

The three side chains attached to the phenyl ring may be connected tothe 1, 2, and 3 positions; the 1, 2, and 4 positions; or the 1, 3 and 5positions of the phenyl ring.

The values for m1, m2 and m3 are each independently 0, 1, 2, 3, 4 or 5.

The values for n1, n2, and n3 are each independently 1, 2, 3, 4 or 5.

X^(1⊖), X^(2⊖) and X^(3⊖) are each independently an organic or inorganicanion.

L¹, L² and L³ are each independently chosen from the group consisting of—CH₂—CH₂—, cis —CH═CH—, trans —CH═CH—, —C≡C—, —CH₂—S—, —S—CH₂—,—Se—CH₂—, —CH₂—Se—, —CH₂—O—, —O—CH₂—, —CH₂—NH—, —NH—CH₂—, —CH₂—NR— whereR is a branched or straight chain alkyl group of one to four carbons,—NR—CH₂— where R is a branched or straight chain alkyl group of one tofour carbons, —CH═N—, —N═CH—, and —═N—.

R¹, R², and R³ are each independently five or six membered nitrogencontaining rings as shown in formulas (IIA) and (IIB).

A¹ is carbon or nitrogen, provided that when A¹ joins a ring atom withan unsaturated bond or is a nitrogen, R⁹ is absent, and when A¹ joins aring atom with an unsaturated bond and is a nitrogen, both R⁴ and R⁹ areabsent.

A² is carbon or nitrogen, provided that when A² joins a ring atom withan unsaturated bond or is a nitrogen, R¹⁰ is absent, and when A² joins aring atom with an unsaturated bond and is a nitrogen, both R⁵ and R¹⁰are absent.

A³ is carbon or nitrogen, provided that when A³ joins a ring atom withan unsaturated bond or is a nitrogen, R¹¹ is absent, and when A³ joins aring atom with an unsaturated bond and is a nitrogen, both R⁶ and R¹¹are absent.

A⁴ is carbon or nitrogen, provided that when A⁴ joins a ring atom withan unsaturated bond or is a nitrogen, R¹² is absent, and when A⁴ joins aring atom with an unsaturated bond and is a nitrogen, both R⁷ and R¹²are absent.

A⁵ is carbon or nitrogen, provided that when A⁵ joins a ring atom withan unsaturated bond or is a nitrogen, R¹³ is absent, and when A⁵ joins aring atom with an unsaturated bond and is a nitrogen, both R⁸ and R¹³are absent.

A⁶ is carbon or nitrogen, provided that when A⁶ joins a ring atom withan unsaturated bond or is a nitrogen, R¹⁹ is absent, and when A⁶ joins aring atom with an unsaturated bond and is a nitrogen, both R¹⁵ and R¹⁹are absent.

A⁷ is carbon or nitrogen, provided that when A⁷ joins a ring atom withan unsaturated bond or is a nitrogen, R²⁰ is absent, and when A⁷ joins aring atom with an unsaturated bond and is a nitrogen, both R¹⁶ and R²⁰are absent.

A⁸ is carbon or nitrogen, provided that when A⁸ joins a ring atom withan unsaturated bond or is a nitrogen, R²¹ is absent, and when A⁸ joins aring atom with an unsaturated bond and is a nitrogen, both R¹⁷ and R²¹are absent.

A⁹ is carbon or nitrogen, provided that when A⁹ joins a ring atom withan unsaturated bond or is a nitrogen, R²² is absent, and when A⁹ joins aring atom with an unsaturated bond and is a nitrogen, both R¹⁸ and R²²are absent.

R¹⁴ or R²³ is absent when any of the bonds to the ammonium nitrogen isunsaturated, and R¹⁴ or R²³ is a straight chain or branched alkyl groupof four carbons or fewer when all of the bonds to the ammonium nitrogenare saturated.

R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹³ or R¹⁵, R¹⁶, R¹⁷, R¹⁸,R¹⁹, R²⁰, R²¹, and R²², when present, are each independently selectedfrom hydrogen, alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,aryl, substituted aryl, alkylaryl, substituted alkylaryl, arylalkyl,substituted arylalkyl, arylalkenyl, substituted arylalkenyl,arylalkynyl, substituted arylalkynyl, heterocyclic, substitutedheterocyclic, halo, cyano, nitro, SOY¹, SO₂Y¹, SO₂OY¹ or SO₂NHY¹, whereY¹ is selected from hydrogen, lower alkyl, alkenyl, alkynyl or aryl, andwhere Y¹ is not hydrogen in SOY¹ and if Y¹ is alkenyl or alkynyl, thesite of unsaturation is not conjugated with a heteroatom; COY², where Y²is selected from hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkoxy, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, alkylaryl, substitutedalkylaryl, arylalkyl, substituted arylalkyl, arylalkenyl, substitutedarylalkenyl, arylalkynyl, substituted arylalkynyl, heterocyclic, orsubstituted heterocyclic, and where if Y² comprises alkenyl or alkynyl,the site of unsaturation is not conjugated with the carbonyl group; OY³,where Y³ is selected from hydrogen, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, alkylaryl,substituted alkylaryl, arylalkyl, substituted arylalkyl, arylalkenyl,substituted arylalkenyl, arylalkynyl, substituted arylalkynyl, acyl,substituted acyl, alkylsulfonyl, arylsulfonyl, heterocyclic, orsubstituted heterocyclic, where if Y³ comprises alkenyl or alkynyl, thesite of unsaturation is not conjugated with the oxygen; NY⁴Y⁵, where Y⁴and Y⁵ are each independently selected from hydrogen, alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, alkylaryl,substituted alkylaryl, arylalkyl, substituted arylalkyl, arylalkenyl,substituted arylalkenyl, arylalkynyl, substituted arylalkynyl, acyl,substituted acyl, alkylsulfonyl, arylsulfonyl, heterocyclic, orsubstituted heterocyclic, where if Y⁴ or Y⁵ comprises alkenyl oralkynyl, the site of unsaturation is not conjugated with the nitrogen;SY⁶, where Y⁶ is selected from hydrogen, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, alkylaryl,substituted alkylaryl, arylalkyl, substituted arylalkyl, arylalkenyl,substituted arylalkenyl, arylalkynyl, substituted arylalkynyl,heterocyclic, or substituted heterocyclic, and where if Y⁶ comprisesalkenyl or alkynyl, the site of unsaturation is not conjugated with thesulfur; or R⁴ and R⁵ together with A¹ and A², or R⁵ and R⁶ together withA² and A³, or R¹⁵ and R¹⁶ together with A⁶ and A⁷, or R¹⁶ and R¹⁷together with A⁷ and A⁸ independently form a three to eight membercyclolkane, substituted cycloalkane, cycloalkene, substitutedcycloalkene, aryl, substituted aryl, heterocycle with one to threehetero atoms in the ring, or substituted heterocycle with one to threehetero atoms in the ring.

For example, R¹, R², and R³ include pyrrole, pyrrolidine, pyrazole,imidazole, 1,2,3-triazole, 1,2,4-triazole, pyridine, piperidine,quinoline, tetrahydroquinoline, isoquinoline, tetrahydroisoquinoline,pyrazine, piperazine, pyridazine, and triazine.

As another example, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁵,R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, and R²², include hydrogen, methyl, ethyl,propyl, butyl, trifluoromethyl, pyrrolidine, N-alkyl pyrrolidine (forexample where the alkyl chain is methyl, ethyl or propyl), unsaturatedpyrrolidine, unsaturated N-alkyl pyrrolidine (for example where thealkyl chain is methyl, ethyl or propyl), aziridine, N-methyl aziridine,azetidine, N-methyl azetidine, unsaturated azetidine, unsaturatedN-methyl azetidine, piperidine, N-methyl piperidine, unsaturatedpiperidine, unsaturated N-methyl piperidine, azepane, N-methyl azepane,unsaturated azepane, unsaturated N-methyl azepane, azocane, N-methylazocane, unsaturated azocane, unsaturated N-methyl azocane,1-aza-bicyclo[3.2.1]octane, 1-aza-bicyclo[2.2.1]heptane,8-methyl-8-aza-bicyclo [3.2.1]octane,1-aza-tricyclo[3.3.1.1^(3,7)]decane, methyl cycloalkyl, methylsubstituted cycloalkyl, methylpyrrolidine, methyl N-alkyl pyrrolidine(for example where the alkyl chain is methyl, ethyl or propyl), methylunsaturated pyrrolidine, methyl unsaturated N-alkyl pyrrolidine (forexample where the alkyl chain is methyl, ethyl or propyl), methylaziridine, methyl N-methyl aziridine, methyl azetidine, methyl N-methylazetidine, methyl unsaturated azetidine, methyl unsaturated N-methylazetidine, methyl piperidine, methyl N-methyl piperidine, methylunsaturated piperidine, methyl unsaturated N-methyl piperidine, methylazepane, methyl N-methyl azepane, methyl unsaturated azepane, methylunsaturated N-methyl azepane, methyl azocane, methyl N-methyl azocane,methyl unsaturated azocane, methyl unsaturated N-methyl azocane,methyl-1-aza-bicyclo[3.2.1]octane, methyl-1-aza-bicyclo[2.2.1]heptane,8-methyl-8-aza-bicyclo[3.2.1]octane, andmethyl-1-aza-tricyclo[3.3.1.1^(3,7)]decane.

As a further example, when R⁴ and R⁵ together with A¹ and A², or R⁵ andR⁶ together with A² and A³, or R¹⁵ and R¹⁶ together with A⁶ and A⁷, orR¹⁶ and R¹⁷ together with A⁷ and A⁸ independently form a three toeight-membered ring, that ring may be a heterocycle containing up tothree hetero atoms (for example nitrogen, oxygen or sulfur) in the ring,and further may be substituted with one or more substituents. Forexample, possible rings include benzene, pyridine, pyran, indene,isoindene, benzofuran, isobenzofuran, benzo[b]thiophene,benzo[c]thiophene, indole, indolenine, isoindole,cyclopental[b]pyridine, pyrano[3,4-b]pynrole, indazole, indoxazine,benzoxazole, anthranil naphthalene, tetralin, decalin, chromene,coumarin, chroman-4-one, isocoumarin, isochromen-3-one, quinoline,isoquinoline, cinnoline, quinazoline, naphthyrdine,pyrido[3,4-b]-pyridine, pyridol[3,2-b]pyridine, pyrido[4,3,-b]-pyridine,benzoxazine, anthracene, phenanthrene, phenalene, fluorene, carazole,xanthene, acnidine, octahydro-[1]pyridine,1-methyloctahydro-[1]pyridine, octahydroindole,1-methyloctahydro-indole, octahydro-cyclopenta[b]pyrrole,1-methyloctahydro-cyclopenta[b]pyrrole, decahydroquinoline, and1-methyldecahydroquinoline.

X^(1⊖), X^(2⊖), and X^(3⊖), for example, include F⁻, Cl⁻, Br⁻, I⁻, NO₂⁻, HSO₄ ⁻, SO₄ ⁻, HPO₄ ⁻, PO₄ ²⁻, methanesulfonate, trifluoromethanesulfate, p-toluenesulfonate, benzenesulfonate, salicylate, proprionate,ascorbate, aspartate, fumarate, galactarate, maleate, citrate,glutamate, glycolate, lactate, malate, maleate, tartrate, oxalate,succinate, or similar pharmaceutically acceptable organic acid additionsalts, including the pharmaceutically acceptable salts listed in theJournal of Pharmaceutical Sciences volume 66, page 2, 1977, which arehereby incorporated by reference. The above salt forms may be in somecases hydrates or solvates with alcohols and other solvents.

In a compound of Formula (I), preferably the phenyl ring is substitutedat the 1, 3 and 5 positions.

In a compound of Formula (I), preferably A¹, A², A³, A⁴, and A⁵ arecarbon. In a compound of Formula (I), preferably R¹, R², and R³ aresubstituted, six-membered, aromatic rings. More preferably, R¹, R², andR³ are substituted pyridinium rings.

In a compound of Formula (I), preferably R⁴ is hydrogen, alkyl, or formsan aryl ring with A¹, A² and R⁵. More preferably, R⁴ is hydrogen, methylor forms a phenyl group with A¹, A² and R⁵.

In a compound of Formula (I), preferably R⁵ is hydrogen, alkyl, phenyl,1-methyl-2-pyrrolidinyl, forms a six-membered ring with A¹, A² and R⁴,or forms an aryl ring with A², A³ and R⁶. More preferably, R⁵ ishydrogen, methyl, butyl, phenyl, 1-methyl-2-pyrrolidinyl, forms a phenylgroup with A¹, A² and R⁴, or forms a phenyl group with A², A³ and R⁶.

In a compound of Formula (I), preferably R⁶ is hydrogen, alkyl, or formsan aryl ring with A², A³ and R⁵. More preferably, R⁶ is hydrogen, methylor forms a phenyl group with A², A³ and R⁵.

In a compound of Formula (I), preferably R⁷ is hydrogen or alkyl. Morepreferably, R⁷ is hydrogen or methyl.

In a compound of Formula (I), preferably R⁵ is hydrogen.

In a compound of Formula (I), preferably m=0.

In a compound of Formula (I), preferably n=3.

In a compound of Formula (I), preferably L¹, L² and L³ are —CH₂—CH₂— or—C≡C—.

In a compound of Formula (I), preferably X^(1⊖), X^(2⊖), and X^(3⊖) arehalogens. More preferably, X^(1⊖), X^(2⊖), and X^(3⊖) are bromide.

In one embodiment, the compound of Formula (I) is defined wherein thephenyl ring is 1,3,5 substituted; wherein m=0; wherein n=3; wherein L is—CH₂CH₂— or —C≡C—; wherein R¹, R², and R³ are pyridinium rings; whereinR⁴ is hydrogen, methyl or forms a phenyl group with A¹, A² and R⁵;wherein R⁵ is hydrogen, methyl, phenyl, butyl, 1-methyl-2-pyrrolidinyl,forms a phenyl group with A¹, A² and R⁴, or forms a phenyl group withA², A³ and R⁶; wherein R⁶ is hydrogen, methyl or forms a phenyl groupwith A², A³ and R⁵; and wherein X¹, X², and X³ are Br.

In another embodiment, the compound of Formula (I) is defined whereinthe phenyl ring is 1,3,5 substituted; wherein m=0; wherein n=3; whereinL is —CH₂CH₂—; wherein R¹, R², and R³ are pyridinium rings; wherein R⁴is hydrogen, methyl or forms a phenyl group with A¹, A² and R⁵; whereinR⁵ is hydrogen, methyl, phenyl, butyl, 1-methyl-2-pyrrolidinyl, forms aphenyl group with A¹, A² and R⁴, or forms a phenyl group with A², A³ andR⁶; wherein R⁶ is hydrogen, methyl or forms a phenyl group with A², A³and R⁵; and wherein X¹, X², and X³ are Br.

In another embodiment, the compound of Formula (I) is defined whereinthe phenyl ring is 1,3,5 substituted; wherein in =0; wherein n=3;wherein L is —C≡C—; wherein R¹, R², and R³ are pyridinium rings; whereinR⁴ is hydrogen, methyl or forms a phenyl group with A¹, A² and R⁵;wherein R⁵ is hydrogen, methyl, phenyl, butyl, 1-methyl-2-pyrrolidinyl,forms a phenyl group with A¹, A² and R⁴, or forms a phenyl group withA², A³ and R⁶; wherein R⁶ is hydrogen, methyl or forms a phenyl groupwith A², A³ and R⁵; and wherein X¹, X², and X³ are Br.

Exemplary compounds for this application are presented in Table 1.

TABLE 1

ID # R4 R5 R6 R7 L GZ550A hydrogen methyl hydrogen hydrogen —C≡C— GZ550Bhydrogen phenyl hydrogen hydrogen —C≡C— GZ551A hydrogen butyl hydrogenhydrogen —C≡C— GZ551B hydrogen 1-methyl-2-pyrrolidinyl hydrogen hydrogen—C≡C— GZ552A phenyl with R⁵ phenyl with R⁴ hydrogen hydrogen —C≡C—GZ552B hydrogen phenyl with R⁶ phenyl with R⁵ hydrogen —C≡C— GZ553Ahydrogen methyl hydrogen methyl —C≡C— GZ553B hydrogen methyl methylhydrogen —C≡C— GZ554A methyl hydrogen hydrogen ' hydrogen —C≡C— GZ554Bhydrogen hydrogen methyl hydrogen —C≡C— GZ555A hydrogen methyl hydrogenhydrogen —CH₂CH₂— GZ555B methyl hydrogen hydrogen hydrogen —CH₂CH₂—GZ555C hydrogen hydrogen methyl hydrogen —CH₂CH₂— GZ556A phenyl with R⁵phenyl with R⁴ hydrogen hydrogen —CH₂CH₂— GZ556B hydrogen phenyl with R⁶phenyl with R⁵ hydrogen —CH₂CH₂— GZ557A hydrogen methyl hydrogen methyl—CH₂CH₂— GZ557B hydrogen methyl methyl hydrogen —CH₂CH₂— GZ558A hydrogenphenyl hydrogen hydrogen —CH₂CH₂— GZ558B hydrogen butyl hydrogenhydrogen —CH₂CH₂— GZ558C hydrogen 1-methyl-2-pyrrolidinyl hydrogenhydrogen —CH₂CH₂—

Exemplary compounds of the present invention include:1,3,5-tris-[5-(2-picolinium)-pent-1-ynyl]-benzene tribromide;1,3,5-tris-[5-(3-picolinium)-pent-1-ynyl]-benzene tribromide;1,3,5-tris-[5-(4-picolinium)-pent-1-ynyl]-benzene tribromide;1,3,5-tris-[5-(3-butyl-pyridinium)-pent-1-ynyl]-benzene tribromide;1,3,5-tris-[5-(3-phenyl-pyridinium)-pent-1-ynyl]-benzene tribromide;1,3,5-tris-{5-[3-(1-methyl-2-S-pyrrolidinyl)pyridinium]-pent-1-ynyl}-benzenetribromide; 1,3,5-tris-[5-(1-quinolinium)-pent-1-ynyl]-benzenetribromide; 1,3,5-tris-[5-(2-isoquinolinium)-pent-1-ynyl]-benzenetribromide; 1,3,5-tris-[5-(3,5-lutidinium)-pent-1-ynyl]-benzenetribromide; 1,3,5-tris-[5-(3,4-lutidinium)-pent-1-ynyl]-benzenetribromide; 1,3,5-tris-[5-(2-picolinium)-pentyl]-benzene tribromide;1,3,5-tris-[5-(3-picolinium)-pentyl]benzene tribromide;1,3,5-tris-[5-(4-picolinium)-pentyl]-benzene tribromide;1,3,5-tris-[5-(3-butyl-pyridinium)-pentyl]-benzene tribromide;1,3,5-tris-[5-(3-phenyl-pyridinium)-pentyl]benzene tribromide;1,3,5-tris-{5-[3-(1-methyl-2-S-pyrrolidinyl)pyridinium]-pentyl}-benzenetribromide; 1,3,5-tris-[5-(1-quinolinium)-pentyl]-benzene tribromide;1,3,5-tris-[5-(2-isoquinolinium)-pentyl]-benzene tribromide;1,3,5-tris-[5-(3,5-lutidinium)-pentyl]-benzene tribromide; and1,3,5-tris-[5-(3,4-lutidinium)-pentyl]-benzene tribromide.

The compounds of the present invention may contain one or morestereocenters. The invention includes all possible diastereomers and allenantiomeric forms as well as racemic mixtures. The compounds can beseparated into substantially optically pure compounds.

The compounds of the invention are nicotinic acetylcholine receptoragents. Thus, they may augment or inhibit [³H]nicotine binding, [³H]MLAbinding, evoke or inhibit neurotransmitter release, and/or evoke orinhibit the flux of ions through the nicotinic receptor. Moreover, thecompounds of the invention may act either at presynaptic sites orpostsynaptic sites, for example, at a postsynaptic acetylcholinereceptor containing an α7 subunit. When acting at a postsynaptic site,neurotransmitter release per se is not altered. Rather, the compounds ofthe invention may act by interacting with a postsynaptic acetylcholinereceptor to change the membrane potential of the cell, therebyincreasing or decreasing the likelihood of firing an action potential.Alternatively, interaction of a compound of the invention with apostsynaptic acetylcholine receptor may result in the alteration of oneor more second messenger systems within the cell so as to decrease orincrease the nicotinic cholinergic response.

In one embodiment, the present invention relates to a method forselectively modulating the function of a nicotinic acetylcholinereceptor comprising administering to a mammalian subject in need thereofa therapeutically effective amount of a compound of Formula (I). In sucha method, the compound of Formula (I) may selectively bind to one ormore subtypes of nicotinic acetylcholine receptor. The compound ofFormula (I) may act as an agoinst or partial agonist of nicotinicacetylcholine receptor function. Hence the compound of Formula (I) mayincrease or prolong the release of a neurotransmitter from a centralnervous system tissue. The neurotransmitter affected may includedopamine, norepinephrine, serotonin, gamma-aminobutryic acid, orglutamate. Alternatively, the compound of Formula (I) may act as anantagonist of nicotinic acetylcholine receptor function. Hence thecompound of Formula (I) may decrease the extent or duration of therelease of a neurotransmitter from a central nervous system tissue. Inthis regard, the compound of Formula (I) may act by decreasingstimulant-evoked neurotransmitter release. The neurotransmitter affectedmay include dopamine, norepinephrine, serotonin, gamma-aminobutryicacid, or glutamate. Alternatively, the compound of Formula (I) may actby interacting with a postsynaptic acetylcholine receptor to change themembrane potential of the cell thereby increasing or decreasing thelikelihood of firing an action potential, or to alter one or more secondmessenger systems within the cell so as to decrease or increase thenicotinic cholinergic response.

In another embodiment, the present invention is directed to a method forpreventing and/or treating a central nervous system associated disordercomprising administering to a mammalian subject in need thereof atherapeutically effective amount of a compound of Formula (I). In such amethod, the compound of Formula (I) may selectively bind to one or moresubtypes of nicotinic acetylcholine receptor. The compound of Formula(I) may act as an agoinst or partial agonist of nicotinic acetylcholinereceptor function. Hence the compound of Formula (I) may increase orprolong the release of a neurotransmitter from a central nervous systemtissue. The neurotransmitter affected may include dopamine,norepinephrine, serotonin, gamma-aminobutryic acid, or glutamate.Alternatively, the compound of Formula (I) may act as an antagonist ofnicotinic acetylcholine receptor function. Hence the compound of Formula(I) may decrease the extent or duration of the release of aneurotransmitter from a central nervous system tissue. In this regard,the compound of Formula (I) may act by decreasing stimulant-evokedneurotransmitter release. The neurotransmitter affected may includedopamine, norepinephrine, serotonin, gamma-aminobutryic acid, orglutamate. Alternatively, the compound of Formula (I) may act byinteracting with a postsynaptic acetylcholine receptor to change themembrane potential of the cell thereby increasing or decreasing thelikelihood of firing an action potential, or to alter one or more secondmessenger systems within the cell so as to decrease or increase thenicotinic cholinergic response.

Central nervous system disorders which may be treated according to themethod of the present invention include Alzheimer's disease, dementia,cognitive dysfunctions (including disorders of attention, focus andconcentration), attention deficit disorders, affective disorders,extrapyramidal motor function disorders, Parkinson's disease,progressive supramolecular palsy, Huntington's disease, Gilles de laTourette syndrome, tardive dyskinesia, neuroendocrine disorders,dysregulation of food intake, disorders of nociception, pain, mood andemotional disorders, depression, panic anxiety, psychosis,schizophrenia, or epilepsy.

In yet another embodiment, the present invention is directed to a methodfor preventing and/or treating substance use and/or abuse comprisingadministering to a mammalian subject in need thereof a therapeuticallyeffective amount of a compound of Formula (I). In such a method, thecompound of Formula (I) may selectively bind to one or more subtypes ofnicotinic acetylcholine receptor. The compound of Formula (I) may act asan agoinst or partial agonist of nicotinic acetylcholine receptorfunction. Hence the compound of Formula (I) may increase or prolong therelease of a neurotransmitter from a central nervous system tissue. Theneurotransmitter affected may include dopamine, norepinephrine,serotonin, gamma-aminobutryic acid, or glutamate. Alternatively, thecompound of Formula (I) may act as an antagonist of nicotinicacetylcholine receptor function. Hence the compound of Formula (I) maydecrease the extent or duration of the release of a neurotransmitterfrom a central nervous system tissue. In this regard, the compound ofFormula (I) may act by decreasing stimulant-evoked neurotransmitterrelease. The neurotransmitter affected may include dopamine,norepinephrine, serotonin, gamma-aminobutryic acid, or glutamate.Alternatively, the compound of Formula (I) may act by interacting with apostsynaptic acetylcholine receptor to change the membrane potential ofthe cell thereby increasing or decreasing the likelihood of firing anaction potential, or to alter one or more second messenger systemswithin the cell so as to decrease or increase the nicotinic cholinergicresponse.

The conditions of substance use and/or abuse treated according to themethod of the present invention include nicotine abuse (including use insmoking cessation therapy), nicotine intoxication, amphetamine abuse,methamphetamine abuse, MDMA (methylenedioxymethamphetamine) abuse,methylphenidate abuse, cocaine abuse, or alcohol abuse.

In another embodiment, the present invention is directed to a method forpreventing and/or treating gastrointestinal tract disorders comprisingadministering to a mammalian subject in need thereof a therapeuticallyeffective amount of a compound of Formula (I). In such a method, thecompound of Formula (I) may selectively bind to one or more subtypes ofnicotinic acetylcholine receptor. The compound of Formula (I) may act asan agonist or partial agonist of nicotinic acetylcholine receptorfunction. Hence the compound of Formula (I) may increase or prolong therelease of a neurotransmitter from a central nervous system tissue, ormay increase or prolong the release of a neurotransmitter from aperipheral nervous system tissue, or may act directly on agastrointestinal tract tissue. The neurotransmitter affected may includedopamine, norepinephrine, serotonin, gamma-aminobutryic acid, orglutamate. Alternatively, the compound of Formula (I) may act as anantagonist of nicotinic acetylcholine receptor function. Hence thecompound of Formula (I) may decrease the extent or duration of therelease of a neurotransmitter from a central nervous system tissue, ormay decrease the extent or duration of the release of a neurotransmitterfrom a peripheral nervous system tissue, or may act directly on agastrointestinal tract tissue. In this regard, the compound of Formula(I) may act by decreasing stimulant-evoked neurotransmitter release. Theneurotransmitter affected may include dopamine, norepinephrine,serotonin, gamma-aminobutryic acid, or glutamate. Alternatively, thecompound of Formula (I) may act by interacting with a postsynapticacetylcholine receptor to change the membrane potential of the cellthereby increasing or decreasing the likelihood of firing an actionpotential, or to alter one or more second messenger systems within thecell so as to decrease or increase the nicotinic cholinergic response.

Gastrointestinal disorders which may be treated according to the methodof the present invention include irritable bowel syndrome, colitis,diarrhea, constipation, gastric acid secretion or ulcers.

The compounds of the present invention can be delivered directly or inpharmaceutical compositions along with suitable carriers or excipients,as is well known in the art. For example, a pharmaceutical compositionof the invention may include a conventional additive, such as astabilizer, buffer, salt, preservative, filler, flavor enhancer and thelike, as known to those skilled in the art. Exemplary buffers includephosphates, carbonates, citrates and the like. Exemplary preservativesinclude EDTA, EGTA, BHA, BHT and the like.

An effective amount of such agents can readily be determined by routineexperimentation, as can the most effective and convenient route ofadministration and the most appropriate formulation. Variousformulations and drug delivery systems are available in the art. See,e.g., Gennaro, A. R., ed. (1995) Remington's Pharmaceutical Sciences.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, nasal, or intestinal administration and parenteraldelivery, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, or intraocular injections. Inaddition, the agent or composition thereof may be administeredsublingually or via a spray. The agent or composition thereof may beadministered in a local rather than a systemic manner. For example, asuitable agent can be delivered via injection or in a targeted drugdelivery system, such as a depot or sustained release formulation.

The pharmaceutical compositions of the present invention may bemanufactured by any of the methods well-known in the art, such as byconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Asnoted above, the compositions of the present invention can include oneor more physiologically acceptable carriers such as excipients andauxiliaries that facilitate processing of active molecules intopreparations for pharmaceutical use.

Proper formulation is dependent upon the route of administration chosen.For injection, for example, the composition may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Fortransmucosal or nasal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art. In a preferred embodiment of the presentinvention, the present compounds are prepared in a formulation intendedfor oral administration. For oral administration, the compounds can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject. The compoundsmay also be formulated in rectal compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides.

Pharmaceutical preparations for oral use can be obtained as solidexcipients, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate. Also, wetting agents such as sodium dodecyl sulfate may beincluded.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations for oral administration include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

In one embodiment, the compounds of the present invention can beadministered transdermally, such as through a skin patch, or topically.In one aspect, the transdermal or topical formulations of the presentinvention can additionally comprise one or multiple penetrationenhancers or other effectors, including agents that enhance migration ofthe delivered compound. Transdermal or topical administration could bepreferred, for example, in situations in which location specificdelivery is desired.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orany other suitable gas. In the case of a pressurized aerosol, theappropriate dosage unit may be determined by providing a valve todeliver a metered amount. Capsules and cartridges of, for example,gelatin, for use in an inhaler or insufflator may be formulated. Thesetypically contain a powder mix of the compound and a suitable powderbase such as lactose or starch.

Compositions formulated for parenteral administration by injection,e.g., by bolus injection or continuous infusion can be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Formulations for parenteral administration include aqueoussolutions or other compositions in water-soluble form.

Suspensions of the active compounds may also be prepared as appropriateoily injection suspensions. Suitable lipophilic solvents or vehiclesinclude fatty oils such as sesame oil and synthetic fatty acid esters,such as ethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

As mentioned above, the compositions of the present invention may alsobe formulated as a depot preparation. Such long acting formulations maybe administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thepresent compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Suitable carriers for the hydrophobic molecules of the invention arewell known in the art and include co-solvent systems comprising, forexample, benzyl alcohol, a nonpolar surfactant, a water-miscible organicpolymer, and an aqueous phase. The co-solvent system may be the VPDco-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v ofthe nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol300, made up to volume in absolute ethanol. The VPD co-solvent system(VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in watersolution. This co-solvent system is effective in dissolving hydrophobiccompounds and produces low toxicity upon systemic administration.Naturally, the proportions of a co-solvent system may be variedconsiderably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied. For example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80, the fraction size of polyethyleneglycol may be varied, other biocompatible polymers may replacepolyethylene glycol, e.g., polyvinyl pyrrolidone, and other sugars orpolysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic molecules may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Liposomal delivery systemsare discussed above in the context of gene-delivery systems. Certainorganic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, thecompounds may be delivered using sustained-release systems, such assemi-permeable matrices of solid hydrophobic polymers containing theeffective amount of the composition to be administered. Varioussustained-release materials are established and available to those ofskill in the art. Sustained-release capsules may, depending on theirchemical nature, release the compounds for a few weeks up to over 100days. Depending on the chemical nature and the biological stability ofthe therapeutic reagent, additional strategies for stabilization may beemployed.

For any composition used in the present methods of treatment, atherapeutically effective dose can be estimated initially using avariety of techniques well known in the art. For example, in a cellculture assay, a dose can be formulated in animal models to achieve acirculating concentration range that includes the IC₅₀ as determined incell culture. Dosage ranges appropriate for human subjects can bedetermined, for example, using data obtained from cell culture assaysand other animal studies.

A therapeutically effective dose of an agent refers to that amount ofthe agent that results in amelioration of symptoms or a prolongation ofsurvival in a subject. Toxicity and therapeutic efficacy of suchmolecules can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., by determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratio oftoxic to therapeutic effects is the therapeutic index, which can beexpressed as the ratio LD₅₀/ED₅₀. Agents that exhibit high therapeuticindices are preferred.

Dosages preferably fall within a range of circulating concentrationsthat includes the ED₅₀ with little or no toxicity. Dosages may varywithin this range depending upon the dosage form employed and the routeof administration utilized. The exact formulation, route ofadministration, and dosage should be chosen, according to methods knownin the art, in view of the specifics of a subject's condition.

The amount of agent or composition administered will, of course, bedependent on a variety of factors, including the sex, age, and weight ofthe subject being treated, the severity of the affliction, the manner ofadministration, and the judgment of the prescribing physician. Thepresent compositions may, if desired, be presented in a pack ordispenser device containing one or more unit dosage forms containing theactive ingredient. Such a pack or device may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.Compositions comprising a compound of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein,and are specifically contemplated.

EXAMPLES

The invention is further understood by reference to the followingexamples, which are intended to be purely exemplary of the invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods that are functionally equivalent arewithin the scope of the invention. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications fall within the scope of the appended claims.

Example 1 Preparation of 1,3,5-tris-(5-hydroxypent-1-ynyl)-benzene

1,3,5-Tribromobenzene (10 g, 31.76 mmol), 4-pentyn-1-ol (10.69 g, 127.06mmol) and bis(triphenylphosphine)palladium(II) dichloride were stirredin triethylamine under nitrogen for 5 minutes. Copper(I) iodide (92 mg,0.48 mmol) was added and the mixture was stirred for 6 hours at 80° C.The mixture was cooled to room temperature, filtered through a celitepad and rinsed with ethyl acetate. The combined filtrate was evaporatedto dryness under reduced pressure. The resulting residue was purified bycolumn chromatography (CHCl₃:MeOH 10:1) to afford 7.61 g of1,3,5-tris-(5-hydroxy-1-pentynyl)-benzene. Yield: 74%. ¹H NMR (300 MHz,CDCl₃) δ 7.31 (3, 3h), 3.81 (t, J=6.0 Hz, 6H), 2.52 (t, J=6.9 Hz, 6H),1.85 (m, 6H); ¹³C NMR (75 MHz, CDCl₃) δ 133.8, 124.2, 90.5, 80.0, 61.9,31.5, 16.2 ppm.

Example 2 Preparation of 1,3,5-tris-(5-bromopent-1-ynyl)-benzene

1,3,5-tris-(5-hydroxy-1-pentynyl)-benzene (1.86 g, 5.73 mmol) and carbontetrabromide (7.41 g, 22.35 mmol) were dissolved in dry methylenechloride (40 mL) and cooled to 0° C. Triphenyl phosphine (6.16 g, 23.47mmol) was added dropwide and the mixture was stirred at 0° C. for 30minutes. The mixture was poured into hexanes (200 mL), filtered througha short silica gel column and washed with ethyl acetate/hexanes (1/4).The combined organic solvents were evaporated to dryness under reducedpressure. The resulting residue was purified by column chromatography(hexanes:ethyl acetate 10:1) to afford 2.63 g of1,3,5-tris-(5-bromopent-1-ynyl)-benzene. Yield 89%. ¹H NMR (300 MHz,CDCl₃) δ 7.33 (s, 3H), 3.57 (t, J=6.3 Hz), 2.60 (t, J=6.9 Hz, 6H), 2.12(m, 6H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 133.9, 124.1, 89.2, 80.4, 32.6,31.7, 18.4 ppm.

Example 3 Preparation of 1,3,5-tris-(5-hydroxypentyl)-benzene

1,3,5-tris-(5-hydroxy-1-pentynyl)-benzene (2.84 g, 8.6 mmol) wasdissolved in methanol (30 mL) and 10% Pd/C (5% w/w) was added. Theresulting mixture was hydrogenated on a Parr hydrogenation apparatus (45psi) for 4 hours. The catalyst was removed by filtration through acelite pad. The filter cake was rinsed with methanol, and the combinedorganic liquors were concentrated under reduced pressure. The crudeproduct was purified by column chromatography (CHCl₃:MeOH 6:1) to afford2.84 g of 1,3,5-tris-(5-hydroxypentyl)-benzene. Yield 96%. ¹H NMR (300MHz, CDCl₃) δ 6.81 (s, 3H), 3.62 (t, J=6.3 Hz, 6H), 2.57 (t, J=7.5 Hz,6H), 1.53-1.70 (m, 12H), 1.38 (m, 6H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ142.5, 126.1, 63.1, 36.1, 32.9, 31.5, 25.7 ppm.

Example 4 Preparation of 1,3,5-tris-(5-bromopentyl)-benzene

Preparation of 1,3,5-tris-(5-bromopentyl)-benzene

1,3,5-tris-(5-hydroxypentyl)-benzene (2.83 g, 8.41 mmol) and carbontetrabromide (10.99 g, 32.80 mmol) were dissolved in dry methylenechloride (50 mL) and cooled to 0° C. Triphenyl phosphine (9.03 g, 34.33mmol) was added dropwise and the mixture was stirred for 30 minutes at0° C. The mixture was poured into hexanes (250 mL), filtered through ashort silica gel column and washed with ethyl acetate/hexanes (1/4). Thecombined organic solvents were evaporated to dryness under reducedpressure. The resulting residue was purified by column chromatography(hexanes:ethyl acetate 8:1) to afford 4.08 g of1,3,5-tris-(5-bromopentyl)-benzene. Yield 92%. ¹H NMR (300 MHz, CDCl₃) δ6.81 (s, 3h), 3.41 (t, J=6.9 Hz, 6H), 2.60 (t, J=7.5 Hz, 6H), 1.88 (m,6H), 1.45 (m, 6H) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 142.4, 126.1, 35.9,34.2, 32.9, 30.9, 28.2 ppm.

Example 5 Preparation of1,3,5-tris-[5-(2-picolinium)-pent-1-ynyl]-benzene tribromide

A mixture of 1,3,5-tris-(5-bromopent-1-ynyl)-benzene (223 mg, 0.43 mmol)and 2-picoline (607 mg, 6.52 mmol) was heated at 60-70° C. for 12 hours.The resultant mixture was washed with diethyl ether and then dissolvedin water (15 mL), the aqueous solution was washed with diethyl ether,then lyophilized to afford 327 mg of1,3,5-tris-[5-(2-picolinium)-pent-1-ynyl]-benzene tribromide. Yield 95%.¹H NMR (300 MHz, CD₃OD) δ 9.01 (dd, J=6.3, 0.9 Hz, 3H), 8.44 (dt, J=7.8,1.5 Hz, 3H), 7.92-8.07 (m, 6H), 7.41 (s, 3H), 4.81 (t, J=6.0 Hz), 2.98(s, 9H), 2.70 (t, J=7.2 Hz, 6H), 2.29 (m, 6H) ppm; ¹³C NMR (75 MHz,CD₃OD) δ 157.1, 146.8, 146.6, 134.9, 131.6, 127.1, 125.4, 90.1, 81.6,58.4, 29.9, 20.7, 17.3 ppm.

Example 6 Preparation of1,3,5-tris-[5-(3-picolinium)-pent-1-ynyl]-benzene tribromide

A mixture of 1,3,5-tris-(5-bromopent-1-ynyl)-benzene (256 mg, 0.50 mmol)and 3-picoline (690 mg, 7.50 mmol) was heated at 60-70° C. for 12 hours.The resultant mixture was washed with diethyl ether (30 mL×3) and thendissolved in water (15 mL), the aqueous solution was washed with diethylether, then lyophilized to afford 345 mg of1,3,5-tris-[5-(3-picolinium)-pent-1-ynyl]-benzene tribromide. Yield 87%.¹H NMR (300 MHz, CD₃OD) δ 9.02 (s, 3H), 8.92 (d, J=6.0 Hz, 3H), 8.4 (d,J=8.4 Hz, 3H), 8.01 (dd, J=8.1, 6.0 Hz, 3H), 7.35 (s, 3H), 4.81 (t,J=7.2 Hz, 6H), 2.64 (t, J=6.9 Hz, 6H), 2.58 (s, 9H), 2.35 (m, 6H) ppm;¹³C NMR (75 MHz, CD₃OD) δ 147.5, 145.8, 143.4, 141.3, 134.9, 128.8,125.3, 89.9, 81.5, 62.2, 31.0, 18.7, 17.2 ppm.

Example 7 Preparation of1,3,5-tris-[5-(4-picolinium)-pent-1-ynyl]-benzene tribromide

A mixture of 1,3,5-tris-(5-bromopent-1-ynyl)-benzene (210 mg, 0.41 mmol)and 4-picoline (572 mg, 6.14 mmol) was heated at 60-70° C. for 12 hours.The resultant mixture was washed with diethyl ether and then dissolvedin water (15 mL), the aqueous solution was washed with diethyl ether (30mL×3), then lyophilized to afford 316 mg of1,3,5-tris-[5-(4-picolinium)-pent-1-ynyl]-benzene tribromide. Yield 97%.¹H NMR (300 MHz, CD₃OD) 8.91 (dd, J=5.1, 1.8 Hz, 6H), 7.94 (d, J=6.3 Hz,6H), 4.77 (t, J=6.9 Hz, 6H), 2.63 (t, J=6.6 Hz, 6H), 2.62 (s, 9H), 2.33(m, 6H) ppm; ¹³C NMR (75 MHz, CD₃OD) δ 161.5, 145.2, 134.9, 130.0,125.4, 90.0, 81.4, 61.6, 30.8, 22.2, 17.2 ppm.

Example 8 Preparation of1,3,5-tris-[5-(3-butyl-pyridinium)-pent-1-ynyl]-benzene tribromide

A mixture of 1,3,5-tris-(5-bromopent-1-ynyl)-benzene (240 mg, 0.47 mmol)and 3-butyl pyridine (950 mg, 7.05 mmol) was heated at 60-70° C. for 12hours. The resultant mixture was washed with diethyl ether and thendissolved in water (15 mL), the aqueous solution was washed with diethylether (30 mL×3), then lyophilized to afford 254 mg of1,3,5-tris-[5-(3-butyl-pyridinium)-pent-1-ynyl]-benzene tribromide.Yield 59%. ¹H NMR (300 MHz, CD₃OD) δ 9.04 (s, 3H), 8.94 (d, J=6.0 Hz,3H), 8.44 (d, J=8.1 Hz, 3H), 8.04 (dd, J=8.1, 6.0 Hz, 3H), 7.33 (s, 3H),4.82 (t, J=7.2 Hz, 6H), 2.87 (t, J=7.8 Hz, 6H), 2.63 (t, J=6.9 Hz, 6H),2.35 (m, 6H), 1.69 (m, 6H), 1.42 (m, 6H), 0.97 (t, J=7.5 Hz, 9H) ppm;¹³C NMR (75 MHz, CD₃OD) δ 146.8, 145.8, 145.6, 143.6, 135.0, 129.0,125.3, 89.9, 81.5, 62.2, 33.8, 33.5, 30.9, 23.5, 17.2, 14.3 ppm.

Example 9 Preparation of1,3,5-tris-[5-(3-phenyl-pyridinium)-pent-1-ynyl]-benzene tribromide

A mixture of 1,3,5-tris-(5-bromopent-1-ynyl)-benzene (259 mg, 0.50 mmol)and 3-phenyl pyridine (1.18 g, 7.50 mmol) was heated at 60-70° C. for 12hours. The resultant mixture was washed with diethyl ether and thendissolved in water (15 mL), the aqueous solution was washed with diethylether (30 mL×5), then lyophilized to afford 415 mg of1,3,5-tris-[5-(3-phenyl-pyridinium)-pent-1-ynyl]-benzene tribromide.Yield 85%. ¹H NMR (300 MHz, CD₃OD) δ 9.50 (s, 3H), 9.09 (d, J=6.0 Hz,3H), 8.78 (d, J=8.1 Hz, 3H), 8.16 (dd, J=8.1, 6.0 Hz, 3H), 7.75-7.87 (m,6H), 7.42-7.65 (m, 9H), 7.17 (s, 3H), 4.96 (t, J=6.9 Hz, 6H), 2.69 (t,J=6.3 Hz, 6H), 2.42 (m, 6H) ppm; ¹³C NMR (75 MHz, CD₃OD) δ 144.4, 144.3,144.2, 142.6, 134.9, 134.5, 131.5, 130.8, 129.5, 128.7, 125.1, 90.0,81.5, 62.6, 30.8, 17.4 ppm.

Example 10 Preparation of1,3,5-tris-{5-[3-(1-methyl-2-S-pyrrolidinyl)pyridinium]-pent-1-ynyl}-benzenetribromide

A mixture of 1,3,5-tris-(5-bromopent-1-ynyl)-benzene (246 mg, 0.48 mmol)and S-nicotine (1.5 mL) was heated at 60-70° C. for 12 hours. Theresultant mixture was washed with diethyl ether and then dissolved inwater (15 mL), the aqueous solution was washed with diethyl ether (30mL×5), then lyophilized to afford 440 mg of1,3,5-tris-{5-[3-(1-methyl-2-S-pyrrolidinyl)pyridinium]-pent-1-ynyl}-benzenetribromide. Yield 92%. ¹H NMR (300 MHz, CD₃OD) δ 9.16 (s, 3H), 9.02 (d,J=6.0 Hz, 3H), 8.60 (d, j=8.1 Hz, 3H), 8.12 (dd, J=8,1, 6.0 Hz, 3H),7.40 (s, 3H), 4.86 (t, J=6.9 Hz, 6H), 3.69 (m, 3H), 3.32 (m, 6H),2.14-2.70 (m, 12H), 2.33 (s, 9H), 1.73-2.14 (m, 10H) ppm; ¹³C NMR (75MHz, CD₃OD) δ 146.2, 145.4, 145.0, 135.1, 129.5, 125.3, 89.8, 81.8,68.8, 62.3, 58.0, 40.8, 36.0, 30.8, 24.1, 17.1 ppm.

Example 11 Preparation of1,3,5-tris-[5-(1-quinolinium)-pent-1-ynyl]-benzene tribromide

A mixture of 1,3,5-tris-(5-bromopent-1-ynyl)-benzene (232 mg, 0.45 mmol)and quinoline (880 mg, 6.75 mmol) was heated at 60-70° C. for 12 hours.The resultant mixture was washed with diethyl ether and then dissolvedin water (15 mL), the aqueous solution was washed with diethyl ether (30mL×5), then lyophilized to afford 234 mg of1,3,5-tris-[5-(1-quinolinium)-pent-1-ynyl]-benzene tribromide. Yield58%. ¹H NMR (300 MHz, CD₃OD) δ 9.58 (dd, J=6.0, 4.5 Hz, 3H), 9.18 (d,J=8.1 Hz, 3H), 8.67, d, J=9.0 Hz, 3H), 8.41 (dd, J=8.1, 1.8 Hz, 3H),8.31 (m, 3H), 7.98-8.18 (m, 6H), 7.11 (s, 3H), 132 (t, J=6.9 Hz, 6H),2.75 (t, J=6.6 Hz, 6H), 2.46 (m, 6H) ppm; ¹³C NMR (75 MHz, CD₃OD) δ150.9, 149.3, 139.6, 137.4, 134.8, 132.3, 131.8, 131.4, 125.2, 123.2,119.8, 90.2, 81.4, 58.8, 29.6, 17.5 ppm.

Example 12 Preparation of1,3,5-tris-[5-(2-isoquinolinium)-pent-1-ynyl]-benzene tribromide

A mixture of 1,3,5-tris-(5-bromopent-1-ynyl)-benzene (222 mg, 0.43 mmol)and isoquinoline (840 mg, 6.45 mmol) was heated at 60-70° C. for 12hours. The resultant mixture was washed with diethyl ether and thendissolved in water (15 mL), the aqueous solution was washed with diethylether (30 mL×5), then lyophilized to afford 357 mg of1,3,5-tris-[5-(2-isoquinolinium)-pent-1-ynyl]-benzene tribromide. Yield92%. ¹H NMR (300 MHz, CD₃OD) δ 10.15 (s, 3H), 8.79 (dd, J=6.9, 1.2 Hz,3H), 8.67, d, J=9.0 Hz, 3H), 8.50 (m, 3H), 8.20 (m, 6H), 8.06 (m, 3H),6.54 (s, 3H), 5.00 (t, J=6.9 Hz, 6H), 2.75 (t, J=6.3 Hz, 6H), 2.47 (m,6H) ppm; ¹³C NMR (75 MHz, CD₃OD) δ 151.5, 139.2, 138.5, 136.0, 134.1,132.6, 131.6, 129.2, 128.4, 127.5, 124.7, 90.0, 81.2, 62.6, 30.5, 17.6ppm.

Example 13 Preparation of1,3,5-tris-[5-(3,5-lutidinium)-pent-1-ynyl]-benzene tribromide

A mixture of 1,3,5-tris-(5-bromopent-1-ynyl)-benzene (252 mg, 0.49 mmol)and 3,5-lutidine (790 mg, 7.35 mmol) was heated at 60-70° C. for 12hours. The resultant mixture was washed with diethyl ether and thendissolved in water (15 mL), the aqueous solution was washed with diethylether (30 mL×3), then lyophilized to afford 383 mg of1,3,5-tris-[5-(3,5-lutidinium)-pent-1-ynyl]-benzene tribromide. Yield:94%. ¹H NMR (300 MHz, CD₃OD) δ 8.83 (s, 6H), 8.22 (s, 3H), 7.32 (s, 3H),4.75 (t, J=7.2 Hz, 6H), 2.64 (t, J=6.6 Hz, 6H), 2.53 (s, 18H), 2.35 (m,6H) ppm; ¹³C NMR (75 MHz, CD₃OD) δ 148.0, 143.1, 140.4, 134.9, 125.3,90.1, 81.4, 62.0, 30.9, 18.6, 17.3 ppm.

Example 14 Preparation of1,3,5-tris-[5-(3,4-lutidinium)-pent-1-ynyl]-benzene tribromide

A mixture of 1,3,5-tris-(5-bromopent-1-ynyl)-benzene (235 mg, 0.46 mmol)and 3,4-lutidine (740 mg, 6.90 mmol) was heated at 60-70° C. for 12hours. The resultant mixture was washed with diethyl ether and thendissolved in water (15 mL), the aqueous solution was washed with diethylether (30 mL×3), then lyophilized to afford 319 mg of1,3,5-tris-[5-(3,4-lutidinium)-pent-1-ynyl]-benzene tribromide. Yield:83%. ¹H NMR (300 MHz, CD₃OD) δ 8.91 (s, 3H), 8.80 (dd, J=6.0, 0.9 Hz,3H), 7.88 (d, J=6.0 Hz, 3H), 7.21 (s, 3H), 4.75 (t, J=6.9 Hz, 6H), 2.65(t, J=6.6 Hz, 6H), 2.51 (s, 9H), 2.45 (s, 9H), 2.35 (m, 6H) ppm; ¹³C NMR(75 MHz, CD₃OD) δ 160.1, 144.5, 142.9, 139.8, 134.7, 129.6, 125.3, 90.3,81.2, 61.5, 30.7, 20.4, 17.3, 17.2 ppm

Example 15 Preparation of 1,3,5-tris-[5-(2-picolinium)-pentyl]-benzenetribromide

A mixture of 1,3,5-tris-(5-bromopentyl)-benzene (247 mg, 0.47 mmol) and2-picoline (657 mg, 7.05 mmol) was heated at 60-70° C. for 12 hours. Theresultant mixture was washed with diethyl ether and then dissolved inwater (15 mL), the aqueous solution was washed with diethyl ether (30mL×3), then lyophilized to afford 335 mg of1,3,5-tris-[5-(2-picolinium)-pentyl]-benzene tribromide. Yield: 89%. ¹HNMR (300 MHz, CD₃OD) δ 8.92 (dd, J=6.3, 1.5 Hz, 3H), 8.43 (dt, J=7.8,1.5 Hz, 3H), 8.00 (d, J=7.8 Hz, 3H), 7.91 (t, J=6.3 Hz, 3H), 6.89 (s,3H), 4.60 (t, J=7.8 Hz, 6H), 2.90 (s, 9H), 2.61 (t, J=7.5 Hz, 6H), 1.99(m, 6H), 1.72 (m, 6H), 1.51 (m, 6H) ppm; ¹³C NMR (75 MHz, CD₃OD) δ156.7, 146.5, 146.3, 143.4, 131.5, 127.2, 126.9, 59.3, 36.7, 32.2, 31.2,27.1, 20.7 ppm.

Example 16 Preparation of 1,3,5-tris-[5-(3-picolinium)-pentyl]benzenetribromide

A mixture of 1,3,5-tris-(5-bromopentyl)-benzene (266 mg, 0.51 mmol) and3-picoline (712 mg, 7.65 mmol) was heated at 60-70° C. for 12 hours. Theresultant mixture was washed with diethyl ether and then dissolved inwater (15 mL), the aqueous solution was washed with diethyl ether (30mL×3), then lyophilized to afford 335 mg of1,3,5-tris-[5-(2-picolinium)-pentyl]-benzene tribromide. Yield: 88%. ¹HNMR (300 MHz, CD₃OD) δ 8.94 (s, 3H), 8.84 (d, J=6.0 Hz, 3H), 8.42 (d,J=8.4 Hz, 3H), 7.98 (dd, J=8.1, 6.0 Hz, 3H), 6.86 (s, 3H), 4.62 (t,J=7.8 Hz, 6H), 2.59 (s, 9H), 2.57 (t, J=7.5 Hz, 6H), 2.06 (m, 6H), 1.69(m, 6H), 1.43 (m, 6H) ppm; ¹³C NMR (75 MHz, CD₃OD) δ 147.2, 145.5,143.4, 143.0, 141.2, 128.7, 127.2, 62.9, 36.7, 32.5, 32.2, 27.0, 18.7ppm.

Example 17 Preparation of 1,3,5-tris-[5-(4-picolinium)-pentyl]-benzenetribromide

A mixture of 1,3,5-tris-(5-bromopentyl)-benzene (245 mg, 0.47 mmol) and4-picoline (657 mg, 7.05 mmol) was heated at 60-70° C. for 12 hours. Theresultant mixture was washed with diethyl ether and then dissolved inwater (15 mL), the aqueous solution was washed with diethyl ether (30mL×3), then lyophilized to afford 351 mg of1,3,5-tris-[5-(2-picolinium)-pentyl]-benzene tribromide. Yield: 93%. ¹HNMR (300 MHz, CD₃OD) δ 8.87 (d, J=6.6 Hz, 6H), 7.94 (d, J=6.6 Hz, 6H),6.84 (s, 3H), 4.61 (t, J=7.5 Hz, 6H), 2.69 (s, 9H), 2.57 (t, J=7.5 Hz,6H), 2.05 (m, 6H), 1.68 (m, 6H), 1.41 (m, 6H) ppm; ¹³C NMR (75 MHz,CD₃OD) δ 161.0, 144.8, 143.4, 129.9, 127.2, 62.1, 36.6, 32.4, 32.1,26.8, 22.2 ppm.

Example 18 Preparation of1,3,5-tris-[5-(3-butyl-pyridinium)-pentyl]-benzene tribromide

A mixture of 1,3,5-tris-(5-bromopentyl)-benzene (245 mg, 0.47 mmol) and3-butyl-pyridine (265 mg, 1.86 mmol) was dissolved in butanone (5 mL)and heated at reflux for 24 hours. The resultant mixture was washed withdiethyl ether and then dissolved in water (15 mL), the aqueous solutionwas washed with diethyl ether (30 mL×5), then lyophilized to afford 252mg of 1,3,5-tris-[5-(3-butyl-pyridinium)-pentyl]-benzene tribromide.Yield: 55%. ¹H NMR (300 MHz, CD₃OD) δ 9.00 (s, 3H), 8.86 (dd, J=6.3, 0.6Hz, 3H), 8.46 (d, J=7.8 Hz, 3H), 8.01 (dd, J=7.8, 6.3 Hz, 3H), 6.84 (s,3H), 4.64 (t, J=7.5 Hz, 6H), 2.89 (t, J=7.8 Hz, 6H), 2.58 (t, J=6.6 Hz,6H), 2.07 (m, 6H), 1.65-1.80 (m, 12H), 1.35-1.52 (m, 12H), 0.98 (t,J=7.2 Hz, 9H) ppm; ¹³C NMR (75 MHz, CD₃OD) δ 146.6, 145.7, 145.2, 143.4,128.9, 127.2, 62.9, 36.7, 33.9, 33.4, 32.6, 32.2, 27.0, 23.4, 14.3 ppm.

Example 19 Preparation of1,3,5-tris-[5-(3-phenyl-pyridinium)-pentyl]-benzene tribromide

A mixture of 1,3,5-tris-(5-bromopentyl)-benzene (272 mg, 0.52 mmol) and3-phenyl-pyridine (323 mg, 2.08 mmol) was dissolved in butanone (5 mL)and heated at reflux for 24 hours. The resultant mixture was washed withdiethyl ether and then dissolved in water (15 mL), the aqueous solutionwas washed with diethyl ether (30 mL×5), then lyophilized to afford 215mg of 1,3,5-tris-[5-(3-butyl-pyridinium)-pentyl]-benzene tribromide.Yield: 42%. ¹H NMR (300 MHz, CD₃OD) δ 9.39 (s, 3H), 8.98 (d, J=6.0 Hz,3H), 8.85 (ddd, J=6.0, 1.8, 1.2 Hz, 3H), 8.15 (dd, J=8.1, 6.0 Hz, 3H),7.78-7.90 (m, 6H), 7.50-7.65 (m, 9H), 6.82 (s, 3H), 4.74 (t, 7.8 Hz,6H), 2.55 (t, J=7.6 Hz, 6H), 2.11 (m, 6H), 1.69 (m, 6H), 1.45 (m, 6H)ppm; ¹³C NMR (75 MHz, CD₃OD) δ 144.2, 143.9, 143.4, 142.8, 134.6, 131.5,130.8, 129.4, 128.7, 127.2, 63.3, 36.7, 32.7, 32.2, 27.0 ppm.

Example 20 Preparation of1,3,5-tris-{5-[3-(1-methyl-2-S-pyrrodinyl)pyridinium]-pentyl}-benzenetribromide

A mixture of 1,3,5-tris-(5-bromopentyl)-benzene (297 mg, 0.57 mmol) andS-nicotine (1.5 mL) was heated at 60-70° C. for 12 hours. The resultantmixture was washed with diethyl ether and then dissolved in water (15mL), the aqueous solution was washed with diethyl ether (30 mL×5), thenlyophilized to afford 510 mg of1,3,5-tris-{5-[3-(1-methyl-2-S-pyrrolidinyl)pyridinium]-pentyl}-benzenetribromide. Yield 89%. ¹H NMR (300 MHz, CD₃OD) δ 9.08 (s, 3H), 8.94 (d,J=6.0 Hz, 3H), 8.61 (d, J=8.1 Hz, 3H), 8.08 (dd, J=8.1, 6.0 Hz, 3H),6.83 (s, 3H), 4.67 (t, J=7.5 Hz, 6H), 3.68 (t, 7.5 Hz, 3H), 3.37 (m,6H), 2.35-2.65 (m, 12H), 2.32 (s, 9H), 1.75-2.17 (m, 12H), 1.69 (m, 6H),1.43 (m, 6H) ppm; ¹³C NMR (75 MHz, CD₃OD) δ 145.8, 145.1, 144.8, 143.4,129.4, 127.2, 68.8, 63.1, 58.0, 40.7, 36.7, 36.0, 32.6, 32.2, 27.0, 24.0ppm.

Example 21 Preparation of 1,3,5-tris-[5-(1-quinolinium)-pentyl]-benzenetribromide

A mixture of 1,3,5-tris-(5-bromopentyl)-benzene (251 mg, 0.48 mmol) andquinoline (930 mg, 7.20 mmol) was heated at 60-70° C. for 12 hours. Theresultant mixture was washed with diethyl ether and then dissolved inwater (15 mL), the aqueous solution was washed with diethyl ether (30mL×5), then lyophilized to afford 390 mg of1,3,5-tris-[5-(1-quinolinium)-pentyl]-benzene tribromide. Yield 89%. ¹HNMR (300 MHz, CD₃OD) δ 9.46 (dd, J=6.0, 1.5 Hz, 3H), 9.22 (d, J=8.4 Hz,3H), 8.57 (d, J=9.0 Hz, 3H), 8.45 (dd, J=8.4, 1.5 Hz, 3H), 8.30 (m, 3H),8.02-8.14 (m, 6H), 6.8 (s, 3H), 5.11 (t, 7.5 Hz, 6H), 2.56 (t, J=7.5 Hz,6H), 2.14 (m, 6H), 1.69 (m, 6H), 1.52 (m, 6H) ppm; ¹³C NMR (75 MHz,CD₃OD) δ 150.3, 148.9, 143.4, 139.4, 137.3, 132.2, 131.8, 131.4, 127.2,123.1, 119.9, 59.4, 36.7, 32.3, 31.1, 27.3 ppm.

Example 22 Preparation of1,3,5-tris-[5-(2-isoquinolinium)-pentyl]-benzene tribromide

A mixture of 1,3,5-tris-(5-bromopentyl)-benzene (266 mg, 0.51 mmol) andquinoline (988 mg, 7.65 mmol) was heated at 60-70° C. for 12 hours. Theresultant mixture was washed with diethyl ether and then dissolved inwater (15 mL), the aqueous solution was washed with diethyl ether (30mL×5), then lyophilized to afford 410 mg of1,3,5-tris-[5-(2-isoquinolinium)-pentyl]-benzene tribromide. Yield 89%.¹H NMR (300 MHz, CD₃OD) δ 9.99 (s, 3H), 8.69 (dd, J=6.9, 1.5 Hz, 3H),8.47-8.54 (m, 6H), 8.22-8.36 (m, 6H), 8.07 (m, 3H), 4.78 (t, J=7.5 Hz,6H), 2.53 (t, J=7.5 Hz, 6H), 2.15 (m, 6H), 1.67 (m, 6H), 1.44 (m, 6H);¹³C NMR (75 MHz, CD₃OD) δ 150.8, 143.3, 138.8, 138.2, 135.8, 132.5,131.5, 129.0, 128.5, 127.5, 127.1, 62.8, 36.5, 32.3, 32.0, 26.8 ppm.

Example 23 Preparation of 1,3,5-tris-[5-(3,5-lutidinium)-pentyl]-benzenetribromide

A mixture of 1,3,5-tris-(5-bromopentyl)-benzene (251 mg, 0.48 mmol) and3,5-lutidine (770 mg, 7.20 mmol) was heated at 60-70° C. for 12 hours.The resultant mixture was washed with diethyl ether and then dissolvedin water (15 mL), the aqueous solution was washed with diethyl ether (30mL×3), then lyophilized to afford 372 mg of1,3,5-tris-[5-(3,5-lutidinium)-pentyl]-benzene tribromide. Yield 92%. ¹HNMR (300 MHz, CD₃OD) δ 8.75 (s, 6H), 8.26 (s, 3H), 6.84 (s, 3H), 4.56(t, J=7.8 Hz, 6H), 2.58 (t, j=7.8 Hz, 6H), 2.54 (s, 18H), 2.05 (m, 6H),1.69 (m, 6H), 1.43 (m, 6H) ppm; ¹³C NMR (75 MHz, CD₃OD) δ 147.7, 143.4,142.7, 140.4, 127.2, 62.7, 36.7, 32.6, 32.3, 27.1, 18.5 ppm.

Example 24 Preparation of 1,3,5-tris-[5-(3,4-lutidinium)-pentyl]-benzenetribromide

A mixture of 1,3,5-tris-(5-bromopentyl)-benzene (264 mg, 0.50 mmol) and3,4-lutidine (808 mg, 7.50 mmol) was heated at 60-70° C. for 12 hours.The resultant mixture was washed with diethyl ether and then dissolvedin water (15 mL), the aqueous solution was washed with diethyl ether (30mL×3), then lyophilized to afford 370 mg of1,3,5-tris-[5-(3,4-lutidinium)-pentyl]-benzene tribromide. Yield 87%. ¹HNMR (300 MHz, CD₃OD) δ 8.78 (s, 3H), 8.67 (d, J=6.0 Hz, 3H), 7.85 (d,J=6.0 Hz, 3H), 6.83 (s, 3H), 4.53 (t, J=7.8 Hz, 6H), 2.59 (s, 9H), 2.57(t, J=8.1 Hz, 6H), 2.48 (s, 9H), 2.03 (m, 6H), 1.68 (m, 6H), 1.42 (m,6H) ppm; ¹³C NMR (75 MHz, CD₃OD) δ 159.8, 144.1, 143.4, 142.5, 139.9,129.5, 127.2, 62.0, 36.7, 32A, 32.3, 27.0, 20.4, 17.1 ppm.

Example 25 Inhibition of [³H]Nicotine (NIC) Binding Assay

Striata from two rats were dissected, pooled, and homogenized with aTekmar polytron in 10 vol of ice-cold modified Krebs-HEPES buffer (20 mMHEPES, 118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, adjusted topH 7.5). The homogenates were incubated at 37° C. for 5 minutes andcentrifuged at 15,000 g for 20 minutes. The pellet was resuspended in 10volumes of ice-cold MilliQ water, incubated for 5 minutes at 37° C., andcentrifuged at 15,000 g for 20 minutes. The second pellet was thenresuspended in 10 volumes of fresh ice-cold 10% Krebs-HEPES buffer,incubated at 37° C., and centrifuged at 15,000 g for 20 minutes. Thelatter sequence of resuspension, incubation and centrifugation wasrepeated. The pellet was frozen under fresh 10% Krebs-HEPES buffer andstored at −40° C. until assay. Upon assay, the pellet was resuspended inKrebs-HEPES buffer, incubated at 37° C. for 5 minutes, and centrifugedat 15,000 g for 20 minutes. The final pellet was resuspended in 3.6 mLice-cold MilliQ water which provided for approximately 200 μg proteinper 100 μL aliquot. Competition assays were performed in duplicate in afinal volume of 200 μl Krebs-HEPES buffer containing 250 mmol Trisbuffer (pH 7.5 at 4° C.). Reactions were initiated by addition of 100 μLof membrane suspension to 3 nM [³H]NIC (50 μL) and one of at least nineconcentrations of analog (50 μL). After a 90 minute incubation at 4° C.,reactions were terminated by dilution of the samples with 3 mL ofice-cold Krebs-HEPES buffer followed immediately by filtration throughWhatman GF/B glass fiber filters (presoaked in 0.5% polyethyleneimine)using a Brandel Cell Harvester. Filters were rinsed three times with 3mL of ice-cold Krebs-HEPES buffer, transferred to scintillation vials,and 5 mL scintillation cocktail (Research Products International Corp.,Mt. Prospect, Ill.) added. Nonspecific binding determined in triplicatewas defined as binding in the presence of 10 μM NIC. Binding parameterswere determined using the weighted, least squares non-linear regression.The tris-quaternary ammonium salts were evaluated for their ability todisplace [³H]NIC binding from rat striatal membranes. The results aresummarized in Table 1.

Example 26 Inhibition of [³H]Methyllycaconitine (MLA) Binding Assay

Rat brain was dissected into the whole brain tissue without cortex,striatum and cerebellum and was frozen in liquid nitrogen and stored at−70° C. until use. The brain tissue was homogenized with a TekmarPolytron (setting 40) in 20 volumes of ice-cold hypotonic buffer (2 mMHEPES, 14.4 mM NaCl, 0.15 mM KCl, 0.2 mM CaCl₂ and 0.1 mM MgSO₄,pH=7.5). The homogenate was incubated at 37° C. for 10 minutes andcentrifuged at 25,000 g for 15 minutes at 40° C. The pellet was washed 3times more by resuspension in 20 volumes of the same buffer andcentrifugation at the above parameters. The final pellet was stored at−20° C. under 4.6 mL of the incubation buffer and was suspended justbefore the incubation with radioligand.

The binding of [³H]MLA to probe α7-type neuronal nicotinic acetylcholinereceptors was measured using a modification of the method of Davies etal., “Characterisation of the binding of [³H]methyllycaconitine (MLA): aradioligand for labelling α7-type neuronal nicotinic acetylcholinereceptors,” Neuropharmacology, 38, 679-690 (1999). [³H]MLA (25.4Ci/mmol) was purchased from Tocris Cookson Ltd., Bristol, U.K. Bindingwas performed in duplicate, in a final volume of 250 μL of theincubation medium, containing 20 mM HEPES, 144 mM NaCl, 1.5 mM KCl, 2 mMCaCl₂, 1 mM MgSO₄ and 0.05% BSA, pH=7.5. Reaction was initiated by theaddition of 100 μL of membrane suspension to the samples containing adesired concentration of test compounds and 2.5 mM [³H]MLA (finalconcentration) and incubated for 2 hours at room temperature. Totalbinding was measured in the absence of unlabelled ligand, andnonspecific binding was determined by the presence of 1 μM unlabelledMLA. The binding reaction was terminated by dilution of samples with 3mL of ice-cold incubation buffer followed by immediate filtrationthrough glass fiber filters (S&S, grade #32, presoaked in 0.5%polyethylenimine) using a Brandel harvester system. Filters were rinsedthree times with 3 mL of ice-cold buffer, transferred to scintillationvials and 4 mL of scintillation cocktail was added. Protein was measuredusing the Bradford dye-binding procedure with bovine serum albumin asthe standard.

In order to determine if these compounds have selectivity at the α7receptor subtype, the tris-alkyl pryidino analogs were evaluated fortheir ability to displace [³H]MLA binding from rat brain membranes, as aresult of their interaction with the α7 receptor (Table 1). In addition,the classical α7 receptor antagonist α-bungarotoxin was also examined inthis assay for comparison. α-Bungarotoxin afforded a K₁ value of28.6±5.4 nM in this assay. The tris-quaternary ammonium salts wereevaluated for their ability to displace [³H]MLA binding from rat brainmembranes. The results are summarized in Table 1.

Example 27 Inhibition of Nicotine-Evoked [³H]Neurotransmitter ReleaseAssay

Rat striatal slices (500 μm thickness, 6-8 mg wet weight) were incubatedfor 30 minutes in Krebs buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl₂,1.0 mM NaH₂PO₄, 1.3 mM CaCl₂, 11.1 mM glucose, 25 mM NaHCO₃, 0.11 mM:L-ascorbic acid and 0.004 mM disodium EDTA; pH 7.4, and saturated with95% O₂/5% CO₂) in a metabolic shaker at 34° C. Slices were rinsed with15 mL of fresh buffer and incubated for an additional 30 minutes infresh buffer containing 0.1 μM [³H]dopamine (DA; 6 slices/3 mL).Subsequently, slices were rinsed with 15 mL of fresh buffer andtransferred to a glass superfusion chamber. Slices were superfused (1.0mL/min) for 60 minutes with Krebs buffer containing nomifensine (10 μM)and pargyline (10 μM) and maintained at 34° C., pH 7.4 with continualaeration (95% O₂/5% CO₂). Two five minute samples (5 mL each) werecollected to determine basal outflow of [³H]DA. Analogs were added tothe superfusion buffer after the collection of the second sample andremained in the buffer until 12 consecutive five minute samples werecollected. Subsequently, S-(−)-nicotine (10 μM) was added to the bufferand an additional 12 consecutive five minute samples were collected. Atthe end of the experiment, each slice was solubilized and the [³H]content of the tissue determined.

Radioactivity in the superfusate and tissue samples were determined byliquid scintillation spectroscopy. Fractional release for tritiumcollected in each sample was divided by the total tritium present at thetime of sample collection and was expressed as a percentage of totaltritium. Basal [³H] outflow was calculated from the average of thetritium collected in the two five-minute samples just before addition ofthe analog. The sum of the increase in collected tritium resulting fromeither exposure to the test compound or exposure to S-(−)-nicotine inthe absence and presence of the test compound equaled total [³H]overflow. [³H] Overflow was calculated by subtracting the [³H] outflowduring an equivalent period of prestimulation from the values in samplescollected during and after drug exposure. Inasmuch as the radiolabelledcompounds were not separated and identified, the tritium in thesuperfusate is referred to as either [³H] outflow or [³H] overflow,rather than [³H]DA. [³H] Overflow primarily represents [³H]DA in thepresence of nomifesine and pargyline in the superfusion buffer. Theanalogs were evaluated for their ability to evoke [³H]DA release fromrat striatal slices. In addition, the classical competitive nicotinicantagonist DHβE was also examined in this assay for comparison. None ofthe compounds examined had any significant [³H]DA releasing propertiesin this assay in the concentration range tested.

Analogs were also evaluated for their ability to inhibit NIC evoked[³H]DA release. In these experiments, the striatal slices weresuperfused for 60 minutes with 100 nM concentration of the analogs priorto NIC (10 μM) exposure. Antagonist activity was evaluated by comparingthe NIC-evoked [³H] overflow in the absence and presence of the analogs.The relative order of potency of the analogs for inhibition ofNIC-evoked [³H]DA release from rat striatal slices is illustrated inTable 1.

Example 28 Inhibition of ACh Evoked Current Through Expressed NicotinicReceptors

Rat nicotinic receptor cDNAs clones were linearized and purified, andRNA transcripts were prepared in vitro using the appropriate mMessagemMachine kit. Mature (>9 cm) female Xenopus laevis African toads (Nasco,Ft. Atkinson, Wis.), housed in the Division of Lab Animal Resources atthe University of Florida were used as a source of oocytes. Prior tosurgery, frogs were anesthetized by placing the animal in a 1.5 g/Lsolution of MS222 for 30 minutes. Oocytes were removed from an incisionmade in the abdomen. In order to remove the follicular cell layer,harvested oocytes were treated with 1.25 mg/mL collagenase for 2 hoursat room temperature in calcium-free Barth's solution (88 mM NaCl, 1 mMKCl, 2.38 mM NaHCO₃, 0.82 mM MgSO₄, 15 mM HEPES, pH 7.6, 0.1 mg/mLgentamicin sulfate). Subsequently, stage 5 oocytes were isolated andinjected with 5-20 ng in 50 mL each of the appropriate subunit cRNAs.Recordings were made 2 to 15 days after injection.

Electrophysiology experiments were conducted using OpusXpress 6000A(Axon instruments, Union City, Calif.), or manual oocyte two-electrodevoltage-clamp systems as previously reported (Stokes et al., 2004).OpusXpress is an integrated system that provides automated impalementand voltage clamp of up to eight oocytes in parallel. Cells wereautomatically perfused with bath solution, and agonist solutions weredelivered from a 96-well plate. Both the voltage and current electrodeswere filled with 3 M KCl. The agonist solutions were applied viadisposable tips, which eliminated the possibility ofcross-contamination. Fresh ACh stock solutions were made daily inRinger's solution and diluted. Flow rates were set at 2 mL/min forexperiments with α7 nicotinic receptors and 4 mL/min for other nicotinicreceptor subtypes. Cells were voltage-clamped at a holding potential of−60 mV. Data were collected at 50 Hz and filtered at 20 Hz. AChapplications were 12 seconds in duration followed by 181 second washoutperiods with α7 nicotinic receptors and 8 seconds with 241 second washperiods for other nicotinic receptor subtypes. For manual oocyterecordings, Warner Instruments (Hamden, Conn.) OC-725C oocyte aplifierswere used, and data were acquired with a minidigi or digidata 1200A withpClamp9 software (Axon instruments). Sampling rates were between 10 and20 HZ and the data were filtered at 6 Hz. Cells were voltage clamped ata holding potential of −50 mV.

Each oocyte received two initial control applications of ACh, then anicotine or analog application, and then a follow-up control applicationof ACh. The control ACh concentrations for α7 and α4β2 receptors were300 μM and 10 μM each, respectively, and 100 μM for the other subtypecombinations tested. Responses to each nicotine or analog applicationwere calculated relative to the preceding ACh control responses in orderto normalize the data, compensating for the varying levels of channelexpression among the oocytes. Responses to nicotine or analog wereinitially normalized to the ACh control response values and thenadjusted to reflect drug response relative to ACh maximums. Responsesfor α7 receptors were calculated as net charge (Papke et al., 2002). Forsubtypes other than α7, responses were calculated from the peak currentamplitudes. Means and standard errors (SEM) were calculated from thenormalized responses of at least four oocytes for each drugconcentration. Since the application of some concentrations of nicotinecaused the subsequent ACh control responses to be reduced due to someform of residual inhibition (or prolonged desensitization), subsequentcontrol responses were compared to the pre-application control AChresponses. When cells failed to recover to at least 75% of the previouscontrol they were replaced with new cells.

For concentration-response relations, data derived from net-chargeanalyses were plotted using Kaleidagraph 3.0.2 (Abelbeck Software;Reading, Pa.) and curves were generated from the Hill equation

${Response} = \frac{{I_{\max}\lbrack{agonist}\rbrack}^{n}}{\lbrack{agonist}\rbrack^{n} + \left( {EC}_{50} \right)^{n}}$where I_(max) denotes the maximal response for a particularagonist/subunit combination, and represents the Hill coefficient.I_(max), n and the EC₅₀ were all unconstrained for the fitting of thenicotine responses, and I_(max) was constrained to equal 1 for the AChresponses, since we used the maximal ACh responses to define fullagonist activity. Negative Hill slopes were applied for the calculationof IC₅₀ values associated with residual inhibition or desensitizationwhen observed.

Results from these experiments are shown in FIGS. 1-4.

FIG. 1 shows concentration-response curves for GZ551A in each of foursubtypes of nicotinic receptor (α4β2, α3β2, α3β4 and α7) expressed inXenopus oocytes. Data are expressed as mean±standard error of the meanand the X-axis illustrates mM concentration of analog. Theelectrophysiological response to the analog during co-application ofacetylcholine is normalized to the acetylcholine control responseobtained for each oocyte. As such, 1.0 indicates that theelectrophysiological response to the analog plus acetylcholine was notdifferent from the response to acetylcholine alone (control), i.e., 100%response. Normalization provides compensation for the varying levels ofnicotinic receptor expression among the oocytes. Concentration of analogwhich inhibits the response to acetylcholine by 50% (IC50 value) areprovided in the legend for each nicotinic receptor subtype. Generally,GZ551A was a potent and selective inhibitor of the α7 nicotinic receptorsubtype.

FIG. 2 shows concentration-response curves for GZ55113 in each of foursubtypes of nicotinic receptor (α4β2, α3β2, α3β4 and α7) expressed inXenopus oocytes. Data are expressed as mean standard error of the meanand the X-axis illustrates mM concentration of analog. Theelectrophysiological response to the analog during co-application ofacetylcholine is normalized to the acetylcholine control responseobtained for each oocyte. As such, 1.0 indicates that theelectrophysiological response to the analog plus acetylcholine was notdifferent from the response to acetylcholine alone (control), i.e., 100%response. Normalization provides compensation for the varying levels ofnicotinic receptor expression among the oocytes. Concentration of analogwhich inhibits the response to acetylcholine by 50% (IC50 value) areprovided in the legend for each nicotinic receptor subtype. Generally,GZ551B was most potent as an inhibitor of the α7 nicotinic receptorsubtype, but was not selective, since it also potently inhibited theα3β4 nicotinic receptor subtype.

FIG. 3 shows concentration-response curves for GZ558C in each foursubtypes of nicotinic receptor (α4β2, α3β2, α3β4 and α7) expressed inXenopus oocytes. Data are expressed as mean±standard error of the meanand the X-axis illustrates mM concentration of analog. Theelectrophysiological response to the analog during co-application ofacetylcholine is normalized to the acetylcholine control responseobtained for each oocyte. As such, 1.0 indicates that theelectrophysiological response to the analog plus acetylcholine was notdifferent from the response to acetylcholine alone (control), i.e., 100%response. Normalization provides compensation for the varying levels ofnicotinic receptor expression among the oocytes. Concentration of analogwhich inhibits the response to acetylcholine by 50% (IC50 value) areprovided in the legend for each nicotinic receptor subtype. Generally,GZ558C was also a potent and selective inhibitor of the α7 nicotinicreceptor subtype.

FIG. 4 shows the inhibitory response of the α7 nicotinic receptorsubtype expressed in Xenopus oocytes to 1.0 mM analog concentration for17 analogs co-applied with 60 mM acetylcholine (filled bars), as well asrecovery from analog-induced inhibition following washing of thepreparation (open bars). Data are expressed as mean±standard error ofthe mean response for each analog. The electrophysiological response tothe analog during co-application of acetylcholine is normalized to theacetylcholine control response obtained for each oocyte. As such, 1.0indicates that the electrophysiological response to the analog plusacetylcholine was not different from the response to acetylcholine alone(control), i.e., 100% response or no inhibitory activity. Normalizationprovides compensation for the varying levels of nicotinic receptorexpression among the oocytes. Generally, the figure shows thatstructural variation in the analog series has a profound effect withregards to inhibition of the α7 nicotinic receptor subtype. Furthermore,recovery was not complete for one analog, GZ558B.

TABLE 1 tris-Quaternary Ammonium Salts Inhibition of [³H]NIC and [³H]MLABinding to Rat Striatal Nicotinic Receptors Inhibition of Nicotine-Inhibition of Inhibition of evoked [³H]Nicotine [³H]MLA [³H]DA Compoundbinding binding Release GZ-550A 10 ± 1.9% 0 ± 0% 46%

GZ-550B 15 ± 0.9% 39 ± 2.2% 49%

GZ-551A 6.80 ± 2.0% 30 ± 2.0% 52%

GZ-551B 44 ± 3.1% 4.2 ± 2.2% 63%

GZ-552A 7.0 ± 3.8% 21 ± 6.0% 72%

GZ-552B 9.2 ± 5.0% 4.5 ± 2.6% 49%

GZ-553A 8.9 ± 2.9% 0 ± 0% 31%

GZ-553B 4.0 ± 1.4% 1.8 ± 1.8%  0%

GZ-554A 4.5 ± 2.0% 0 ± 0% 68%

GZ-554B 21 ± 4.5% 0 ± 0% 34%

GZ-555A 14 ± 6.1% 1.2 ± 1.2% 42%

GZ-555B 8.6 ± 7.2% 0 ± 0% 25%

GZ-555C 6.0 ± 3.4% 0 ± 0% 37 ± 17%

(n = 4) GZ-556A 5.7 ± 5.7% 6.8 ± 0.9% 8.0 ± 6.4%

(n = 3) GZ-556B 8.4 ± 7.6% 2.5 ± 1.5% 28 ± 11%

(n = 3) GZ-557A 11 ± 2.9% 6.5 ± 3.6% 39 ± 18%

(n = 5) GZ-557B 9.3 ± 4.1% 0.2 ± 0.2% 50 ± 21%

(n = 5) GZ-558A 14 ± 5.8% 19 ± 1.7% 6.0 ± 5.0%

(n = 3) GZ-558B 6.2 ± 4.0% 4.4 ± 3.3% 68 ± 14%

(n = 4) GZ-558C 81 ± 2.3% 7.9 ± 7.1% 35%

Data are % inhibition at 100 nM concentration of analog for at least 1-3independent experiments. Specific binding in the [³H]NIC binding assayis calculated at the difference between the total binding of 3 nM[³H]NIC and nonspecific binding in the presence of 10 μM cold nicotine.Specific binding for the [³H]MLA binding assay is calculated as thedifference between the total binding of 2.5 nM [³H]MLA to the receptorsalone and its nonspecific binding in the presence of 1 μM cold MLA.Analog-induced inhibition of nicotine-evoked [³H]DA release iscalculated as a percent of that in the absence of analog.

It will be appreciated that, although specific embodiments of theinvention have been described herein for purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. All such modifications and variations areintended to be included herein within the scope of this disclosure andthe present invention and protected by the following claims.

We claim:
 1. A method for selectively modulating the function of anicotinic acetylcholine receptor comprising administering to a mammaliansubject in need thereof a therapeutically effective amount of a compoundof Formula (I):

wherein the three side chains attached to the phenyl ring may beconnected to the 1, 2, and 3 positions; the 1, 2, and 4 positions; orthe 1, 3 and 5 positions of the phenyl ring; wherein m1, m2 and m3 areeach independently 0, 1, 2, 3, 4 or 5; wherein n1, n2, and n3 are eachindependently 1, 2, 3, 4, or 5; wherein X^(1⊖), X^(2⊖), and X^(3⊖) areeach independently an organic or inorganic anion; wherein L¹, L² and L³are each independently chosen from the group consisting of —CH₂—CH₂—,cis —CH═CH—, trans —CH═CH—, —C≡C—, —CH₂—S—, —S—CH₂—, —Se—CH₂—, —CH₂—Se—,—CH₂—O—, —O—CH₂—, —CH₂—NH—, —NH—CH₂—, —CH₂—NR— where R is a branched orstraight chain alkyl group of one to four carbons, —NR—CH₂— where R is abranched or straight chain alkyl group of one to four carbons, —CH═N—,—N═CH—, and —N═N—; wherein R¹, R², and R³ are each independently five orsix membered nitrogen containing rings as shown in formulas (IIA) and(IIB)

wherein A¹ is carbon or nitrogen, provided that when A^(l) joins a ringatom with an unsaturated bond or is a nitrogen, R⁹ is absent, and whenA¹ joins a ring atom with an unsaturated bond and is a nitrogen, both R⁴and R⁹ are absent; wherein A² is carbon or nitrogen, provided that whenA² joins a ring atom with an unsaturated bond or is a nitrogen, R¹⁰ isabsent, and when A² joins a ring atom with an unsaturated bond and is anitrogen, both R⁵ and R¹⁰ are absent; wherein A³ is carbon or nitrogen,provided that when A³ joins a ring atom with an unsaturated bond or is anitrogen, R¹¹ is absent, and when A³ joins a ring atom with anunsaturated bond and is a nitrogen, both R⁶ and R¹¹ are absent; whereinA⁴ is carbon or nitrogen, provided that when A⁴ joins a ring atom withan unsaturated bond or is a nitrogen, R¹² is absent, and when A⁴ joins aring atom with an unsaturated bond and is a nitrogen, both R⁷ and R¹²are absent; wherein A⁵ is carbon or nitrogen, provided that when A⁵joins a ring atom with an unsaturated bond or is a nitrogen, R¹³ isabsent, and when A⁵ joins a ring atom with an unsaturated bond and is anitrogen, both R⁸ and R¹³ are absent; wherein A⁶ is carbon or nitrogen,provided that when A⁶ joins a ring atom with an unsaturated bond or is anitrogen, R¹⁹ is absent, and when A⁶ joins a ring atom with anunsaturated bond and is a nitrogen, both R¹⁵ and R¹⁹ are absent; whereinA⁷ is carbon or nitrogen, provided that when A⁷ joins a ring atom withan unsaturated bond or is a nitrogen, R²⁰ is absent, and when A⁷ joins aring atom with an unsaturated bond and is a nitrogen, both R¹⁶ and R²⁰are absent; wherein A⁸ is carbon or nitrogen, provided that when A⁸joins a ring atom with an unsaturated bond or is a nitrogen, R²¹ isabsent, and when A⁸ joins a ring atom with an unsaturated bond and is anitrogen, both R¹⁷ and R²¹ are absent; wherein A⁹ is carbon or nitrogen,provided that when A⁹ joins a ring atom with an unsaturated bond or is anitrogen, R²² is absent, and when A⁹ joins a ring atom with anunsaturated bond and is a nitrogen, both R¹⁸ and R²² are absent; whereinR¹⁴ or R²³ is absent when any of the bonds to the ammonium nitrogen isunsaturated, and R¹⁴ or R²³ is a straight chain or branched alkyl groupof four carbons or fewer when all of the bonds to the ammonium nitrogenare saturated; wherein R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹³ orR¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, and R²², when present, are eachindependently selected from hydrogen; alkyl; substituted alkyl;cycloalkyl; substituted cycloalkyl; alkenyl; substituted alkenyl;alkynyl; substituted alkynyl; aryl; substituted aryl; alkylaryl;substituted alkylaryl; arylalkyl; substituted arylalkyl; arylalkenyl;substituted arylalkenyl; arylalkynyl; substituted arylalkynyl;heterocyclic; substituted heterocyclic; halo; cyano; nitro; SOY¹, SO₂Y¹,SO₂OY¹ or SO₂NHY¹, where Y¹ is selected from hydrogen, lower alkyl,alkenyl, alkynyl or aryl, and where Y¹ is not hydrogen in SOY¹ and if Y¹is alkenyl or alkynyl, the site of unsaturation is not conjugated with aheteroatom; COY², where Y² is selected from hydrogen, alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, alkoxy, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,alkylaryl, substituted alkylaryl, arylalkyl, substituted arylalkyl,arylalkenyl, substituted arylalkenyl, arylalkynyl, substitutedarylalkynyl, heterocyclic, or substituted heterocyclic, and where if Y²comprises alkenyl or alkynyl, the site of unsaturation is not conjugatedwith the carbonyl group; OY³, where Y³ is selected from hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, alkylaryl, substituted alkylaryl, arylalkyl, substitutedarylalkyl, arylalkenyl, substituted arylalkenyl, arylalkynyl,substituted arylalkynyl, acyl, susbstituted acyl, alkylsulfonyl,arylsulfonyl, heterocyclic, or substituted heterocyclic, where if Y³comprises alkenyl or alkynyl, the site of unsaturation is not conjugatedwith the oxygen; NY⁴Y⁵, where Y⁴ and Y⁵ are each independently selectedfrom hydrogen, alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,aryl, substituted aryl, alkylaryl, substituted alkylaryl, arylalkyl,substituted arylalkyl, arylalkenyl, substituted arylalkenyl,arylalkynyl, substituted arylalkynyl, acyl, susbstituted acyl,alkylsulfonyl, arylsulfonyl, heterocyclic, or substituted heterocyclic,where if Y⁴ or Y⁵ comprises alkenyl or alkynyl, the site of unsaturationis not conjugated with the nitrogen; SY⁶, where Y⁶ is selected fromhydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, alkylaryl, substituted alkylaryl, arylalkyl,substituted arylalkyl, arylalkenyl, substituted arylalkenyl,arylalkynyl, substituted arylalkynyl, heterocyclic, or substitutedheterocyclic, and where if Y⁶ comprises alkenyl or alkynyl, the site ofunsaturation is not conjugated with the sulfur; or R⁴ and R⁵ togetherwith A¹ and A², or R⁵ and R⁶ together with A² and A³, or R¹⁵ and R¹⁶together with A⁶ and A⁷, or R¹⁶ and R¹⁷ together with A⁷ and A⁸independently form a three to eight member cyclolkane, substitutedcycloalkane, cycloalkene, substituted cycloalkene, aryl, substitutedaryl, heterocycle with one to three hetero atoms in the ring, orsubstituted heterocycle with one to three hetero atoms in the ring. 2.The method of claim 1, wherein the compound of Formula (I) bindsselectively to one or more subtypes of the nicotinic acetylcholinereceptors.
 3. The method of claim 2, wherein the selective modulationcomprises activation of the function of nicotinic acetylcholinereceptors as an agonist or as a partial agonist.
 4. The method of claim2, wherein the selective modulation comprises inactivation of thefunction of nicotinic acetylcholine receptors as an antagonist.
 5. Themethod of claim 2, wherein there is a decrease in the stimulant-evokedrelease of a neurotransmitter from a central nervous system tissue. 6.The method of claim 2, wherein there is an increase in the release of aneurotransmitter from a central nervous system tissue.
 7. The method ofclaim 5, wherein the neurotransmitter released is selected from thegroup consisting of dopamine, norepinephrine, serotonin,gamma-aminobutyric acid, and glutamate.
 8. The method of claim 6,wherein the neurotransmitter released is selected from the groupconsisting of dopamine, norepinephrine, serotonin, gamma-aminobutyricacid, and glutamate.
 9. The method of claim 1, wherein the compound ofFormula (I) is selected from the group consisting of:1,3,5-tris-[5-(2-picolinium)-pent-1-ynyl]-benzene tribromide;1,3,5-tris-[5-(3-picolinium)-pent-1-ynyl]-benzene tribromide;1,3,5-tris-[5-(4-picolinium)-pent-1-ynyl]-benzene tribromide;1,3,5-tris-[5-(3-butyl-pyridinium)-pent-1-ynyl]-benzene tribromide;1,3,5-tris-[5-(3-phenyl-pyridinium)-pent-1-ynyl]-benzene tribromide;1,3,5-tris-{5-[3-(1-methyl-2-S-pyrrolidinyl)pyridinium]-pent-1-ynyl}-benzenetribromide; 1,3,5-tris-[5-(1-quinolinium)-pent-1-ynyl]-benzenetribromide; 1,3,5-tris-[5-(2-isoquinolinium)-pent-1-ynyl]-benzenetribromide; 1,3,5-tris-[5-(3,5-lutidinium)-pent-1-ynyl]-benzenetribromide; 1,3,5-tris-[5-(3,4-lutidinium)-pent-1-ynyl]-benzenetribromide; 1,3,5-tris-[5-(2-picolinium)-pentyl]-benzene tribromide;1,3,5-tris-[5-(3-picolinium)-pentyl]-benzene tribromide;1,3,5-tris-[5-(4-picolinium)-pentyl]-benzene tribromide;1,3,5-tris-[5-(3-butyl-pyridinium)-pentyl]-benzene tribromide;1,3,5-tris-[5-(3-phenyl-pyridinium)-pentyl]-benzene tribromide;1,3,5-tris-{5-[3-(1-methyl-2-S-pyrrolidinyl) pyridinium]-pentyl}-benzenetribromide; 1,3,5-tris-[5-(1-quinolinium)-pentyl]-benzene tribromide;1,3,5-tris-[5-(2-isoquinolinium)-pentyl]-benzene tribromide;1,3,5-tris-[5-(3,5-lutidinium)-pentyl]-benzene tribromide; and1,3,5-tris-[5-(3,4-lutidinium)-pentyl]-benzene tribromide.